Compositions and methods for modulating angiopoietin-like 3 expression

ABSTRACT

Provided herein are methods, compounds, and compositions for reducing expression of an ANGPTL3 mRNA and protein in an animal. Also provided herein are methods, compounds, and compositions for reducing lipids and/or glucose in an animal. Such methods, compounds, and compositions are useful to treat, prevent, delay, or ameliorate any one or more of cardiovascular disease and/or metabolic disease, or a symptom thereof, in an individual in need thereof.

SEQUENCE LISTING

The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled BIOL0254USC2SEQ_ST25.txt, created on Feb. 26, 2018 which is 0.98 MB in size. The information in the electronic format of the sequence listing is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

Provided herein are methods, compounds, and compositions for reducing expression of angiopoietin-like 3 (ANGPTL3) mRNA and protein in an animal. Also, provided herein are methods, compounds, and compositions having an ANGPTL3 inhibitor for reducing ANGPTL3 related diseases or conditions in an animal. Such methods, compounds, and compositions are useful, for example, to treat, prevent, delay or ameliorate any one or more of cardiovascular disease or metabolic syndrome, or a symptom thereof, in an animal.

BACKGROUND

Diabetes and obesity (sometimes collectively referred to as “diabesity”) are interrelated in that obesity is known to exacerbate the pathology of diabetes and greater than 60% of diabetics are obese. Most human obesity is associated with insulin resistance and leptin resistance. In fact, it has been suggested that obesity may have an even greater impact on insulin action than diabetes itself (Sindelka et al., Physiol Res., 2002, 51, 85-91). Additionally, several compounds on the market for the treatment of diabetes are known to induce weight gain, a very undesirable side effect to the treatment of this disease.

Cardiovascular disease is also interrelated to obesity and diabetes. Cardiovascular disease encompasses a wide variety of etiologies and has an equally wide variety of causative agents and interrelated players. Many causative agents contribute to symptoms such as elevated plasma levels of cholesterol, including non-high density lipoprotein cholesterol (non-HDL-C), as well as other lipid-related disorders. Such lipid-related disorders, generally referred to as dyslipidemia, include hyperlipidemia, hypercholesterolemia and hypertriglyceridemia among other indications. Elevated non-HDL cholesterol is associated with atherogenesis and its sequelae, including cardiovascular diseases such as arteriosclerosis, coronary artery disease, myocardial infarction, ischemic stroke, and other forms of heart disease. These rank as the most prevalent types of illnesses in industrialized countries. Indeed, an estimated 12 million people in the United States suffer with coronary artery disease and about 36 million require treatment for elevated cholesterol levels.

Epidemiological and experimental evidence has shown that high levels of circulating triglyceride (TG) can contribute to cardiovascular disease and a myriad of metabolic disorders (Valdivielso et al., 2009, Atherosclerosis Zhang et al., 2008, Circ Res. 1; 102(2):250-6). TG derived from either exogenous or endogenous sources is incorporated and secreted in chylomicrons from the intestine or in very low density lipoproteins (VLDL) from the liver. Once in circulation, TG is hydrolyzed by lipoprotein lipase (LpL) and the resulting free fatty acids can then be taken up by local tissues and used as an energy source. Due to the profound effect LpL has on plasma TG and metabolism in general, discovering and developing compounds that affect LpL activity are of great interest.

Metabolic syndrome is a combination of medical disorders that increase one's risk for cardiovascular disease and diabetes. The symptoms, including high blood pressure, high triglycerides, decreased HDL and obesity, tend to appear together in some individuals. It affects a large number of people in a clustered fashion. In some studies, the prevalence in the USA is calculated as being up to 25% of the population. Metabolic syndrome is known under various other names, such as (metabolic) syndrome X, insulin resistance syndrome, Reaven's syndrome or CHAOS. With the high prevalence of cardiovascular disorders and metabolic disorders there remains a need for improved approaches to treat these conditions

The angiopoietins are a family of secreted growth factors. Together with their respective endothelium-specific receptors, the angiopoietins play important roles in angiogenesis. One family member, angiopoietin-like 3 (also known as angiopoietin-like protein 3, ANGPT5, ANGPTL3, or angiopoietin 5), is predominantly expressed in the liver, and is thought to play a role in regulating lipid metabolism (Kaplan et al., J. Lipid Res., 2003, 44, 136-143). Genome-wide association scans (GWAS) surveying the genome for common variants associated with plasma concentrations of HDL, LDL and triglyceride found an association between triglycerides and single-nucleotide polymorphisms (SNPs) near ANGPTL3 (Willer et al., Nature Genetics, 2008, 40(2):161-169). Individuals with homozygous ANGPTL3 loss-of-function mutations present with low levels of all atherogenic plasma lipids and lipoproteins, such as total cholesterol (TC) and TG, low density lipoprotein cholesterol (LDL-C), apoliprotein B (apoB), non-HDL-C, as well as HDL-C (Romeo et al. 2009, J Clin Invest, 119(1):70-79; Musunuru et al. 2010 N Engl J Med, 363:2220-2227; Martin-Campos et al. 2012, Clin Chim Acta, 413:552-555; Minicocci et al. 2012, J Clin Endocrinol Metab, 97:e1266-1275; Noto et al. 2012, Arterioscler Thromb Vasc Biol, 32:805-809; Pisciotta et al. 2012, Circulation Cardiovasc Genet, 5:42-50). This clinical phenotype has been termed familial combined hypolipidemia (FHBL2). Despite reduced secretion of VLDL, subjects with FHBL2 do not have increased hepatic fat content. They also appear to have lower plasma glucose and insulin levels, and importantly, both diabetes and cardiovascular disease appear to be absent from these subjects. No adverse clinical phenotypes have been reported to date (Minicocci et al. 2013, J of Lipid Research, 54:3481-3490). Reduction of ANGPTL3 has been shown to lead to a decrease in TG, cholesterol and LDL levels in animal models (U.S. Ser. No. 13/520,997; PCT Publication WO 2011/085271). Mice deficient in ANGPTL3 have very low plasma triglyceride (TG) and cholesterol levels, while overpexpression produces the opposite effects (Koishi et al. 2002; Koster 2005; Fujimoto 2006). Accordingly, the potential role of ANGPTL3 in lipid metabolism makes it an attractive target for therapeutic intervention.

To date, therapeutic strategies to treat cardiometabolic disease by directly targeting ANGPTL3 levels have been limited. ANGPTL3 polypeptide fragments (U.S. Ser. No. 12/128,545), anti-ANGPTL3 antibodies (U.S. Ser. No. 12/001,012) and ANGPTL3 nucleic acid inhibitors including antisense oligonucleotides (U.S. Ser. No. 13/520,997; PCT Publication WO 2011/085271; incorporated by reference herein, in their entirety) have previously been suggested or developed, but none of the compounds directly targeting ANGPTL3 have been approved for treating cardiometabolic disease. Accordingly, there is an unmet need for highly potent and tolerable compounds to inhibit ANGPTL3. The invention disclosed herein relates to the discovery of novel, highly potent inhibitors of ANGPTL3 expression and their use in treatment.

SUMMARY OF THE INVENTION

Provided herein are compositions and methods for modulating expression of ANGPTL3 mRNA and protein. In certain embodiments, the composition is an ANGPTL3 specific inhibitor. In certain embodiments, the ANGPTL3 specific inhibitor decreases expression of ANGPTL3 mRNA and protein.

In certain embodiments, the composition is an ANGPTL3 specific inhibitor. In certain embodiments, the ANGPTL3 specific inhibitor is a nucleic acid. In certain embodiments, the nucleic acid is an antisense compound. In certain embodiments, the antisense compound is a modified oligonucleotide. In certain embodiments, the antisense compound is a modified oligonucleotide with a conjugate group attached.

In certain embodiments, the ANGPTL3 specific inhibitor is a modified oligonucleotide with a conjugate group, wherein the modified oligonucleotide consists of 12 to 30 linked nucleosides and having a nucleobase sequence comprising at least 8, least 9, least 10, least 11, at least 12, least 13, at least 14, at least 15, at least 16, least 17, least 18, least 19, or 20 contiguous nucleobases of the nucleobase sequence of SEQ ID NO: 77.

In certain embodiments, the ANGPTL3 specific inhibitor is a modified oligonucleotide with a conjugate group, wherein the modified oligonucleotide consists of 12 to 30 linked nucleosides and comprising a nucleobase sequence comprising a portion of at least 8 contiguous nucleobases complementary to an equal length portion ofnucleobases 1140-1159 of SEQ ID NO: 1, wherein the nucleobase sequence of the modified oligonucleotide is at least 80% complementary to SEQ ID NO: 1.

In certain embodiments, the ANGPTL3 specific inhibitor is a modified oligonucleotide with a conjugate group, wherein the modified oligonucleotide consists of 12 to 30 linked nucleosides and comprising a nucleobase sequence comprising a portion of at least 8 contiguous nucleobases complementary to an equal length portion of nucleobases 9715-9734 of SEQ ID NO: 2, wherein the nucleobase sequence of the modified oligonucleotide is at least 80% complementary to SEQ ID NO: 2.

In certain embodiments, the ANGPTL3 specific inhibitor is a modified oligonucleotide with a conjugate group, wherein the modified oligonucleotide consists of 20 linked nucleosides and having a nucleobase sequence comprising at least 8 contiguous nucleobases of SEQ ID NO: 77, wherein the modified oligonucleotide comprises: (a) a gap segment consisting of ten linked deoxynucleosides; (b) a 5′ wing segment consisting of five linked nucleosides; (c) a 3′ wing segment consisting of five linked nucleosides; and wherein the gap segment is positioned between the 5′ wing segment and the 3′ wing segment, wherein each nucleoside of each wing segment comprises a 2′-O-methoxyethyl sugar, wherein each internucleoside linkage is a phosphorothioate linkage and wherein each cytosine residue is a 5-methylcytosine.

In certain embodiments, the ANGPTL3 specific inhibitor is a modified oligonucleotide with a conjugate group, wherein the modified oligonucleotide consists of 20 linked nucleosides and having a nucleobase sequence consisting of at least 8 contiguous nucleobases of SEQ ID NO: 77, wherein the modified oligonucleotide consists of: (a) a gap segment consisting often linked deoxynucleosides; (b) a 5′ wing segment consisting of five linked nucleosides; (c) a 3′ wing segment consisting of five linked nucleosides; and wherein the gap segment is positioned between the 5′ wing segment and the 3′ wing segment, wherein each nucleoside of each wing segment comprises a 2′-O-methoxyethyl sugar, wherein each internucleoside linkage is a phosphorothioate linkage and wherein each cytosine residue is a 5-methylcytosine.

In certain embodiments, the present disclosure provides conjugated antisense compounds. In certain embodiments, the present disclosure provides conjugated antisense compounds comprising an antisense oligonucleotide complementary to a nucleic acid transcript. In certain embodiments, the present disclosure provides methods comprising contacting a cell with a conjugated antisense compound comprising an antisense oligonucleotide complementary to a nucleic acid transcript. In certain embodiments, the present disclosure provides methods comprising contacting a cell with a conjugated antisense compound comprising an antisense oligonucleotide and reducing the amount or activity of a nucleic acid transcript in a cell.

The asialoglycoprotein receptor (ASGP-R) has been described previously. See e.g., Park et al., PNAS vol. 102, No. 47, pp 17125-17129 (2005). Such receptors are expressed on liver cells, particularly hepatocytes. Further, it has been shown that compounds comprising clusters of three N-acetylgalactosamine (GalNAc) ligands are capable of binding to the ASGP-R, resulting in uptake of the compound into the cell. See e.g., Khorev et al., Bioorganic and Medicinal Chemistry, 16, 9, pp 5216-5231 (May 2008). Accordingly, conjugates comprising such GalNAc clusters have been used to facilitate uptake of certain compounds into liver cells, specifically hepatocytes. For example it has been shown that certain GalNAc-containing conjugates increase activity of duplex siRNA compounds in liver cells in vivo. In such instances, the GalNAc-containing conjugate is typically attached to the sense strand of the siRNA duplex. Since the sense strand is discarded before the antisense strand ultimately hybridizes with the target nucleic acid, there is little concern that the conjugate will interfere with activity. Typically, the conjugate is attached to the 3′ end of the sense strand of the siRNA. See e.g., U.S. Pat. No. 8,106,022. Certain conjugate groups described herein are more active and/or easier to synthesize than conjugate groups previously described.

In certain embodiments of the present invention, conjugates are attached to single-stranded antisense compounds, including, but not limited to RNase H based antisense compounds and antisense compounds that alter splicing of a pre-mRNA target nucleic acid. In such embodiments, the conjugate should remain attached to the antisense compound long enough to provide benefit (improved uptake into cells) but then should either be cleaved, or otherwise not interfere with the subsequent steps necessary for activity, such as hybridization to a target nucleic acid and interaction with RNase H or enzymes associated with splicing or splice modulation. This balance of properties is more important in the setting of single-stranded antisense compounds than in siRNA compounds, where the conjugate may simply be attached to the sense strand. Disclosed herein are conjugated single-stranded antisense compounds having improved potency in liver cells in vivo compared with the same antisense compound lacking the conjugate. Given the required balance of properties for these compounds such improved potency is surprising.

In certain embodiments, conjugate groups herein comprise a cleavable moiety. As noted, without wishing to be bound by mechanism, it is logical that the conjugate should remain on the compound long enough to provide enhancement in uptake, but after that, it is desirable for some portion or, ideally, all of the conjugate to be cleaved, releasing the parent compound (e.g., antisense compound) in its most active form. In certain embodiments, the cleavable moiety is a cleavable nucleoside. Such embodiments take advantage of endogenous nucleases in the cell by attaching the rest of the conjugate (the cluster) to the antisense oligonucleotide through a nucleoside via one or more cleavable bonds, such as those of a phosphodiester linkage. In certain embodiments, the cluster is bound to the cleavable nucleoside through a phosphodiester linkage. In certain embodiments, the cleavable nucleoside is attached to the antisense oligonucleotide (antisense compound) by a phosphodiester linkage. In certain embodiments, the conjugate group may comprise two or three cleavable nucleosides. In such embodiments, such cleavable nucleosides are linked to one another, to the antisense compound and/or to the cluster via cleavable bonds (such as those of a phosphodiester linkage).

Certain conjugates herein do not comprise a cleavable nucleoside and instead comprise a cleavable bond. It is shown that that sufficient cleavage of the conjugate from the oligonucleotide is provided by at least one bond that is vulnerable to cleavage in the cell (a cleavable bond).

In certain embodiments, conjugated antisense compounds are prodrugs. Such prodrugs are administered to an animal and are ultimately metabolized to a more active form. For example, conjugated antisense compounds are cleaved to remove all or part of the conjugate resulting in the active (or more active) form of the antisense compound lacking all or some of the conjugate.

In certain embodiments, conjugates are attached at the 5′ end of an oligonucleotide. Certain such 5′-conjugates are cleaved more efficiently than counterparts having a similar conjugate group attached at the 3′ end. In certain embodiments, improved activity may correlate with improved cleavage. In certain embodiments, oligonucleotides comprising a conjugate at the 5′ end have greater efficacy than oligonucleotides comprising a conjugate at the 3′ end (see, for example, Examples 56, 81, 83, and 84). Further, 5′-attachment allows simpler oligonucleotide synthesis. Typically, oligonucleotides are synthesized on a solid support in the 3′ to 5′ direction. To make a 3′-conjugated oligonucleotide, typically one attaches a pre-conjugated 3′ nucleoside to the solid support and then builds the oligonucleotide as usual. However, attaching that conjugated nucleoside to the solid support adds complication to the synthesis. Further, using that approach, the conjugate is then present throughout the synthesis of the oligonucleotide and can become degraded during subsequent steps or may limit the sorts of reactions and reagents that can be used. Using the structures and techniques described herein for 5′-conjugated oligonucleotides, one can synthesize the oligonucleotide using standard automated techniques and introduce the conjugate with the final (5′-most) nucleoside or after the oligonucleotide has been cleaved from the solid support.

In view of the art and the present disclosure, one of ordinary skill can easily make any of the conjugates and conjugated oligonucleotides herein. Moreover, synthesis of certain such conjugates and conjugated oligonucleotides disclosed herein is easier and/or requires few steps, and is therefore less expensive than that of conjugates previously disclosed, providing advantages in manufacturing. For example, the synthesis of certain conjugate groups consists of fewer synthetic steps, resulting in increased yield, relative to conjugate groups previously described. Conjugate groups such as GalNAc₃-10 in Example 46 and GalNAc₃-7 in Example 48 are much simpler than previously described conjugates such as those described in U.S. Pat. No. 8,106,022 or 7,262,177 that require assembly of more chemical intermediates. Accordingly, these and other conjugates described herein have advantages over previously described compounds for use with any oligonucleotide, including single-stranded oligonucleotides and either strand of double-stranded oligonucleotides (e.g., siRNA).

Similarly, disclosed herein are conjugate groups having only one or two GalNAc ligands. As shown, such conjugates groups improve activity of antisense compounds. Such compounds are much easier to prepare than conjugates comprising three GalNAc ligands. Conjugate groups comprising one or two GalNAc ligands may be attached to any antisense compounds, including single-stranded oligonucleotides and either strand of double-stranded oligonucleotides (e.g., siRNA).

In certain embodiments, the conjugates herein do not substantially alter certain measures of tolerability. For example, it is shown herein that conjugated antisense compounds are not more immunogenic than unconjugated parent compounds. Since potency is improved, embodiments in which tolerability remains the same (or indeed even if tolerability worsens only slightly compared to the gains in potency) have improved properties for therapy.

In certain embodiments, conjugation allows one to alter antisense compounds in ways that have less attractive consequences in the absence of conjugation. For example, in certain embodiments, replacing one or more phosphorothioate linkages of a fully phosphorothioate antisense compound with phosphodiester linkages results in improvement in some measures of tolerability. For example, in certain instances, such antisense compounds having one or more phosphodiester are less immunogenic than the same compound in which each linkage is a phosphorothioate. However, in certain instances, as shown in Example 26, that same replacement of one or more phosphorothioate linkages with phosphodiester linkages also results in reduced cellular uptake and/or loss in potency. In certain embodiments, conjugated antisense compounds described herein tolerate such change in linkages with little or no loss in uptake and potency when compared to the conjugated full-phosphorothioate counterpart. In fact, in certain embodiments, for example, in Examples 44, 57, 59, and 86, oligonucleotides comprising a conjugate and at least one phosphodiester internucleoside linkage actually exhibit increased potency in vivo even relative to a full phosphorothioate counterpart also comprising the same conjugate. Moreover, since conjugation results in substantial increases in uptake/potency a small loss in that substantial gain may be acceptable to achieve improved tolerability. Accordingly, in certain embodiments, conjugated antisense compounds comprise at least one phosphodiester linkage.

In certain embodiments, conjugation of antisense compounds herein results in increased delivery, uptake and activity in hepatocytes. Thus, more compound is delivered to liver tissue. However, in certain embodiments, that increased delivery alone does not explain the entire increase in activity. In certain such embodiments, more compound enters hepatocytes. In certain embodiments, even that increased hepatocyte uptake does not explain the entire increase in activity. In such embodiments, productive uptake of the conjugated compound is increased. For example, as shown in Example 102, certain embodiments of GalNAc-containing conjugates increase enrichment of antisense oligonucleotides in hepatocytes versus non-parenchymal cells. This enrichment is beneficial for oligonucleotides that target genes that are expressed in hepatocytes.

In certain embodiments, conjugated antisense compounds herein result in reduced kidney exposure. For example, as shown in Example 20, the concentrations of antisense oligonucleotides comprising certain embodiments of GalNAc-containing conjugates are lower in the kidney than that of antisense oligonucleotides lacking a GalNAc-containing conjugate. This has several beneficial therapeutic implications. For therapeutic indications where activity in the kidney is not sought, exposure to kidney risks kidney toxicity without corresponding benefit. Moreover, high concentration in kidney typically results in loss of compound to the urine resulting in faster clearance. Accordingly for non-kidney targets, kidney accumulation is undesired.

In certain embodiments, the present disclosure provides conjugated antisense compounds represented by the formula: A-B—C-D

E-F)_(q)

wherein

A is the antisense oligonucleotide;

B is the cleavable moiety

C is the conjugate linker

D is the branching group

each E is a tether;

each F is a ligand; and

q is an integer between 1 and 5.

In the above diagram and in similar diagrams herein, the branching group “D” branches as many times as is necessary to accommodate the number of (E-F) groups as indicated by “q”. Thus, where q=1, the formula is: A-B—C-D-E-F

where q=2, the formula is:

where q=3, the formula is:

where q=4, the formula is:

where q=5, the formula is:

In certain embodiments, conjugated antisense compounds are provided having the structure:

In certain embodiments, conjugated antisense compounds are provided having the structure:

In certain embodiments, conjugated antisense compounds are provided having the structure:

In certain embodiments, conjugated antisense compounds are provided having the structure:

In embodiments having more than one of a particular variable (e.g., more than one “m” or “n”), unless otherwise indicated, each such particular variable is selected independently. Thus, for a structure having more than one n, each n is selected independently, so they may or may not be the same as one another.

In certain embodiments, the present disclosure provides conjugated antisense compounds represented by the following structure. In certain embodiments, the antisense compound comprises the modified oligonucleotide ISIS 563580 with a 5′-X, wherein X is a conjugate group comprising GalNAc. In certain embodiments, the antisense compound consists of the modified oligonucleotide ISIS 563580 with a 5′-X, wherein X is a conjugate group comprising GalNAc.

In certain embodiments, the present disclosure provides conjugated antisense compounds represented by the following structure. In certain embodiments, the antisense compound comprises the conjugated modified oligonucleotide ISIS 703801. In certain embodiments, the antisense compound consists of the conjugated modified oligonucleotide ISIS 703801.

In certain embodiments, the present disclosure provides conjugated antisense compounds represented by the following structure. In certain embodiments, the antisense compound comprises the conjugated modified oligonucleotide ISIS 703802. In certain embodiments, the antisense compound consists of the conjugated modified oligonucleotide ISIS 703802.

In certain embodiments, the present disclosure provides conjugated antisense compounds represented by the following structure. In certain embodiments, the antisense compound comprises a modified oligonucleotide with the nucleobase sequence of SEQ ID NO: 77 with a 5′-GalNAc with variability in the sugar mods of the wings. In certain embodiments, the antisense compound consists of a modified oligonucleotide with the nucleobase sequence of SEQ ID NO: 77 with a 5′-GalNAc with variability in the sugar mods of the wings.

wherein either R′ is —OCH₂CH₂OCH₃ (MOE) and R² is H; or R¹ and R² together form a bridge, wherein R¹ is —O— and R² is —CH₂—, —CH(CH₃)—, or —CH₂CH₂—, and IV and R² are directly connected such that the resulting bridge is selected from: —O—CH₂—, —O—CH(CH₃)—, and —O—CH₂CH₂—;

and for each pair of R³ and R⁴ on the same ring, independently for each ring: either R³ is selected from H and —OCH₂CH₂OCH₃ and R⁴ is H; or R³ and R⁴ together form a bridge, wherein R³ is —O—, and R⁴ is —CH₂—, —CH(CH₃)—, or —CH₂CH₂— and R³ and R⁴ are directly connected such that the resulting bridge is selected from: —O—CH₂—, —O—CH(CH₃)—, and —O—CH₂CH₂—;

and R⁵ is selected from H and —CH₃;

and Z is selected from S⁻ and O⁻.

Certain embodiments provide a composition comprising a conjugated antisense compound described herein, or a salt thereof, and a pharmaceutically acceptable carrier or diluent.

In certain embodiments, the modulation of ANGPTL3 expression occurs in a cell or tissue. In certain embodiments, the modulations occur in a cell or tissue in an animal. In certain embodiments, the animal is a human. In certain embodiments, the modulation is a reduction in ANGPTL3 mRNA level. In certain embodiments, the modulation is a reduction in ANGPTL3 protein level. In certain embodiments, both ANGPTL3 mRNA and protein levels are reduced. Such reduction may occur in a time-dependent or in a dose-dependent manner.

Certain embodiments provide compositions and methods for use in therapy. Certain embodiments provide compositions and methods for preventing, treating, delaying, slowing the progression and/or ameliorating ANGPTL3 related diseases, disorders, and conditions. In certain embodiments, such diseases, disorders, and conditions are cardiovascular and/or metabolic diseases, disorders, and conditions. In certain embodiments, the compositions and methods for therapy include administering an ANGPTL3 specific inhibitor to an individual in need thereof. In certain embodiments, the ANGPTL3 specific inhibitor is a nucleic acid. In certain embodiments, the nucleic acid is an antisense compound. In certain embodiments, the antisense compound is a modified oligonucleotide. In certain embodiments, the antisense compound is a modified oligonucleotide with a conjugate group attached.

DETAILED DESCRIPTION OF THE INVENTION

It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed. Herein, the use of the singular includes the plural unless specifically stated otherwise. As used herein, the use of “or” means “and/or” unless stated otherwise. Furthermore, the use of the term “including” as well as other forms, such as “includes” and “included”, is not limiting. Also, terms such as “element” or “component” encompass both elements and components comprising one unit and elements and components that comprise more than one subunit, unless specifically stated otherwise.

The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. All documents, or portions of documents, cited in this application, including, but not limited to, patents, patent applications, articles, books, and treatises, are hereby expressly incorporated-by-reference for the portions of the document discussed herein, as well as in their entirety.

Definitions

Unless specific definitions are provided, the nomenclature utilized in connection with, and the procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well known and commonly used in the art. Standard techniques can be used for chemical synthesis, and chemical analysis. Certain such techniques and procedures may be found for example in “Carbohydrate Modifications in Antisense Research” Edited by Sangvi and Cook, American Chemical Society, Washington D.C., 1994; “Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa., 21^(st) edition, 2005; and “Antisense Drug Technology, Principles, Strategies, and Applications” Edited by Stanley T. Crooke, CRC Press, Boca Raton, Fla.; and Sambrook et al., “Molecular Cloning, A laboratory Manual,” 2nd Edition, Cold Spring Harbor Laboratory Press, 1989, which are hereby incorporated by reference for any purpose. Where permitted, all patents, applications, published applications and other publications, GENBANK Accession Numbers and associated sequence information obtainable through databases such as National Center for Biotechnology Information (NCBI) and other data referred to throughout in the disclosure herein are incorporated by reference for the portions of the document discussed herein, as well as in their entirety.

Unless otherwise indicated, the following terms have the following meanings:

As used herein, “nucleoside” means a compound comprising a nucleobase moiety and a sugar moiety. Nucleosides include, but are not limited to, naturally occurring nucleosides (as found in DNA and RNA) and modified nucleosides. Nucleosides may be linked to a phosphate moiety.

As used herein, “chemical modification” means a chemical difference in a compound when compared to a naturally occurring counterpart. Chemical modifications of oligonucleotides include nucleoside modifications (including sugar moiety modifications and nucleobase modifications) and internucleoside linkage modifications. In reference to an oligonucleotide, chemical modification does not include differences only in nucleobase sequence.

As used herein, “furanosyl” means a structure comprising a 5-membered ring comprising four carbon atoms and one oxygen atom.

As used herein, “naturally occurring sugar moiety” means a ribofuranosyl as found in naturally occurring RNA or a deoxyribofuranosyl as found in naturally occurring DNA.

As used herein, “sugar moiety” means a naturally occurring sugar moiety or a modified sugar moiety of a nucleoside.

As used herein, “modified sugar moiety” means a substituted sugar moiety or a sugar surrogate.

As used herein, “substituted sugar moiety” means a furanosyl that is not a naturally occurring sugar moiety. Substituted sugar moieties include, but are not limited to furanosyls comprising substituents at the 2′-position, the 3′-position, the 5′-position and/or the 4′-position. Certain substituted sugar moieties are bicyclic sugar moieties.

As used herein, “2′-substituted sugar moiety” means a furanosyl comprising a substituent at the 2′-position other than H or OH. Unless otherwise indicated, a 2′-substituted sugar moiety is not a bicyclic sugar moiety (i.e., the 2′-substituent of a 2′-substituted sugar moiety does not form a bridge to another atom of the furanosyl ring.

As used herein, “MOE” means —OCH₂CH₂OCH₃.

As used herein, “2′-F nucleoside” refers to a nucleoside comprising a sugar comprising fluorine at the 2′ position. Unless otherwise indicated, the fluorine in a 2′-F nucleoside is in the ribo position (replacing the OH of a natural ribose).

As used herein the term “sugar surrogate” means a structure that does not comprise a furanosyl and that is capable of replacing the naturally occurring sugar moiety of a nucleoside, such that the resulting nucleoside sub-units are capable of linking together and/or linking to other nucleosides to form an oligomeric compound which is capable of hybridizing to a complementary oligomeric compound. Such structures include rings comprising a different number of atoms than furanosyl (e.g., 4, 6, or 7-membered rings); replacement of the oxygen of a furanosyl with a non-oxygen atom (e.g., carbon, sulfur, or nitrogen); or both a change in the number of atoms and a replacement of the oxygen. Such structures may also comprise substitutions corresponding to those described for substituted sugar moieties (e.g., 6-membered carbocyclic bicyclic sugar surrogates optionally comprising additional substituents). Sugar surrogates also include more complex sugar replacements (e.g., the non-ring systems of peptide nucleic acid). Sugar surrogates include without limitation morpholinos, cyclohexenyls and cyclohexitols.

As used herein, “bicyclic sugar moiety” means a modified sugar moiety comprising a 4 to 7 membered ring (including but not limited to a furanosyl) comprising a bridge connecting two atoms of the 4 to 7 membered ring to form a second ring, resulting in a bicyclic structure. In certain embodiments, the 4 to 7 membered ring is a sugar ring. In certain embodiments the 4 to 7 membered ring is a furanosyl. In certain such embodiments, the bridge connects the 2′-carbon and the 4′-carbon of the furanosyl.

As used herein, “nucleotide” means a nucleoside further comprising a phosphate linking group. As used herein, “linked nucleosides” may or may not be linked by phosphate linkages and thus includes, but is not limited to “linked nucleotides.” As used herein, “linked nucleosides” are nucleosides that are connected in a continuous sequence (i.e. no additional nucleosides are present between those that are linked).

As used herein, “nucleobase” means a group of atoms that can be linked to a sugar moiety to create a nucleoside that is capable of incorporation into an oligonucleotide, and wherein the group of atoms is capable of bonding with a complementary naturally occurring nucleobase of another oligonucleotide or nucleic acid. Nucleobases may be naturally occurring or may be modified. “Nucleobase sequence” means the order of contiguous nucleobases independent of any sugar, linkage, or nucleobase modification.

As used herein the terms, “unmodified nucleobase” or “naturally occurring nucleobase” means the naturally occurring heterocyclic nucleobases of RNA or DNA: the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) (including 5-methyl C), and uracil (U).

As used herein, “modified nucleobase” means any nucleobase that is not a naturally occurring nucleobase.

As used herein, “modified nucleoside” means a nucleoside comprising at least one chemical modification compared to naturally occurring RNA or DNA nucleosides. Modified nucleosides comprise a modified sugar moiety and/or a modified nucleobase.

As used herein, “bicyclic nucleoside” or “BNA” means a nucleoside comprising a bicyclic sugar moiety.

As used herein, “constrained ethyl nucleoside” or “cEt” means a nucleoside comprising a bicyclic sugar moiety comprising a 4′-CH(CH₃)—O-2′ bridge.

As used herein, “locked nucleic acid nucleoside” or “LNA” means a nucleoside comprising a bicyclic sugar moiety comprising a 4′-CH₂—O-2′ bridge.

As used herein, “2′-substituted nucleoside” means a nucleoside comprising a substituent at the 2′-position other than H or OH. Unless otherwise indicated, a 2′-substituted nucleoside is not a bicyclic nucleoside.

As used herein, “deoxynucleoside” means a nucleoside comprising 2′-H furanosyl sugar moiety, as found in naturally occurring deoxyribonucleosides (DNA). In certain embodiments, a 2′-deoxynucleoside may comprise a modified nucleobase or may comprise an RNA nucleobase (e.g., uracil).

As used herein, “oligonucleotide” means a compound comprising a plurality of linked nucleosides. In certain embodiments, an oligonucleotide comprises one or more unmodified ribonucleosides (RNA) and/or unmodified deoxyribonucleosides (DNA) and/or one or more modified nucleosides.

As used herein “oligonucleoside” means an oligonucleotide in which none of the internucleoside linkages contains a phosphorus atom. As used herein, oligonucleotides include oligonucleosides.

As used herein, “modified oligonucleotide” means an oligonucleotide comprising at least one modified nucleoside and/or at least one modified internucleoside linkage.

As used herein, “linkage” or “linking group” means a group of atoms that link together two or more other groups of atoms.

As used herein “internucleoside linkage” means a covalent linkage between adjacent nucleosides in an oligonucleotide.

As used herein “naturally occurring internucleoside linkage” means a 3′ to 5′ phosphodiester linkage.

As used herein, “modified internucleoside linkage” means any internucleoside linkage other than a naturally occurring internucleoside linkage.

As used herein, “terminal internucleoside linkage” means the linkage between the last two nucleosides of an oligonucleotide or defined region thereof.

As used herein, “phosphorus linking group” means a linking group comprising a phosphorus atom.

Phosphorus linking groups include without limitation groups having the formula:

wherein:

R_(a) and R are each, independently, O, S, CH₂, NH, or NJ₁ wherein J₁ is C₁-C₆ alkyl or substituted C₁-C₆ alkyl;

R_(b) is O or S;

R_(c) is OH, SH, C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₁-C₆ alkoxy, substituted C₁-C₆ alkoxy, amino or substituted amino; and

J₁ is R_(b) is O or S.

Phosphorus linking groups include without limitation, phosphodiester, phosphorothioate, phosphorodithioate, phosphonate, phosphoramidate, phosphorothioamidate, thionoalkylphosphonate, phosphotriesters, thionoalkylphosphotriester and boranophosphate.

As used herein, “internucleoside phosphorus linking group” means a phosphorus linking group that directly links two nucleosides.

As used herein, “non-internucleoside phosphorus linking group” means a phosphorus linking group that does not directly link two nucleosides. In certain embodiments, a non-internucleoside phosphorus linking group links a nucleoside to a group other than a nucleoside. In certain embodiments, a non-internucleoside phosphorus linking group links two groups, neither of which is a nucleoside.

As used herein, “neutral linking group” means a linking group that is not charged. Neutral linking groups include without limitation phosphotriesters, methylphosphonates, MMI (—CH₂—N(CH₃)—O—), amide-3 (—CH₂—C(═O)—N(H)—), amide-4 (—CH₂—N(H)—C(═O)—), formacetal (—O—CH₂—O—), and thioformacetal (—S—CH₂—O—).

Further neutral linking groups include nonionic linkages comprising siloxane (dialkylsiloxane), carboxylate ester, carboxamide, sulfide, sulfonate ester and amides (See for example: Carbohydrate Modifications in Antisense Research; Y. S. Sanghvi and P. D. Cook Eds. ACS Symposium Series 580; Chapters 3 and 4, (pp. 40-65)). Further neutral linking groups include nonionic linkages comprising mixed N, O, S and CH₂ component parts.

As used herein, “internucleoside neutral linking group” means a neutral linking group that directly links two nucleosides.

As used herein, “non-internucleoside neutral linking group” means a neutral linking group that does not directly link two nucleosides. In certain embodiments, a non-internucleoside neutral linking group links a nucleoside to a group other than a nucleoside. In certain embodiments, a non-internucleoside neutral linking group links two groups, neither of which is a nucleoside.

As used herein, “oligomeric compound” means a polymeric structure comprising two or more sub-structures. In certain embodiments, an oligomeric compound comprises an oligonucleotide. In certain embodiments, an oligomeric compound comprises one or more conjugate groups and/or terminal groups. In certain embodiments, an oligomeric compound consists of an oligonucleotide. Oligomeric compounds also include naturally occurring nucleic acids. In certain embodiments, an oligomeric compound comprises a backbone of one or more linked monomeric subunits where each linked monomeric subunit is directly or indirectly attached to a heterocyclic base moiety. In certain embodiments, oligomeric compounds may also include monomeric subunits that are not linked to a heterocyclic base moiety, thereby providing abasic sites. In certain embodiments, the linkages joining the monomeric subunits, the sugar moieties or surrogates and the heterocyclic base moieties can be independently modified. In certain embodiments, the linkage-sugar unit, which may or may not include a heterocyclic base, may be substituted with a mimetic such as the monomers in peptide nucleic acids.

As used herein, “terminal group” means one or more atom attached to either, or both, the 3′ end or the 5′ end of an oligonucleotide. In certain embodiments a terminal group is a conjugate group. In certain embodiments, a terminal group comprises one or more terminal group nucleosides.

As used herein, “conjugate” or “conjugate group” means an atom or group of atoms bound to an oligonucleotide or oligomeric compound. In general, conjugate groups modify one or more properties of the compound to which they are attached, including, but not limited to pharmacodynamic, pharmacokinetic, binding, absorption, cellular distribution, cellular uptake, charge and/or clearance properties.

As used herein, “conjugate linker” or “linker” in the context of a conjugate group means a portion of a conjugate group comprising any atom or group of atoms and which covalently link (1) an oligonucleotide to another portion of the conjugate group or (2) two or more portions of the conjugate group.

Conjugate groups are shown herein as radicals, providing a bond for forming covalent attachment to an oligomeric compound such as an antisense oligonucleotide. In certain embodiments, the point of attachment on the oligomeric compound is the 3′-oxygen atom of the 3′-hydroxyl group of the 3′ terminal nucleoside of the oligomeric compound. In certain embodiments the point of attachment on the oligomeric compound is the 5′-oxygen atom of the 5′-hydroxyl group of the 5′ terminal nucleoside of the oligomeric compound. In certain embodiments, the bond for forming attachment to the oligomeric compound is a cleavable bond. In certain such embodiments, such cleavable bond constitutes all or part of a cleavable moiety.

In certain embodiments, conjugate groups comprise a cleavable moiety (e.g., a cleavable bond or cleavable nucleoside) and a carbohydrate cluster portion, such as a GalNAc cluster portion. Such carbohydrate cluster portion comprises: a targeting moiety and, optionally, a conjugate linker. In certain embodiments, the carbohydrate cluster portion is identified by the number and identity of the ligand. For example, in certain embodiments, the carbohydrate cluster portion comprises 3 GalNAc groups and is designated “GalNAc₃”. In certain embodiments, the carbohydrate cluster portion comprises 4 GalNAc groups and is designated “GalNAc₄”. Specific carbohydrate cluster portions (having specific tether, branching and conjugate linker groups) are described herein and designated by Roman numeral followed by subscript “a”. Accordingly “GalNac3-1_(a)” refers to a specific carbohydrate cluster portion of a conjugate group having 3 GalNac groups and specifically identified tether, branching and linking groups. Such carbohydrate cluster fragment is attached to an oligomeric compound via a cleavable moiety, such as a cleavable bond or cleavable nucleoside.

As used herein, “cleavable moiety” means a bond or group that is capable of being split under physiological conditions. In certain embodiments, a cleavable moiety is cleaved inside a cell or sub-cellular compartments, such as a lysosome. In certain embodiments, a cleavable moiety is cleaved by endogenous enzymes, such as nucleases. In certain embodiments, a cleavable moiety comprises a group of atoms having one, two, three, four, or more than four cleavable bonds.

As used herein, “cleavable bond” means any chemical bond capable of being split. In certain embodiments, a cleavable bond is selected from among: an amide, a polyamide, an ester, an ether, one or both esters of a phosphodiester, a phosphate ester, a carbamate, a di-sulfide, or a peptide.

As used herein, “carbohydrate cluster” means a compound having one or more carbohydrate residues attached to a scaffold or linker group. (see, e.g., Maier et al., “Synthesis of Antisense Oligonucleotides Conjugated to a Multivalent Carbohydrate Cluster for Cellular Targeting,” Bioconjugate Chemistry, 2003, (14): 18-29, which is incorporated herein by reference in its entirety, or Rensen et al., “Design and Synthesis of Novel N-Acetylgalactosamine-Terminated Glycolipids for Targeting of Lipoproteins to the Hepatic Asiaglycoprotein Receptor,” J. Med. Chem. 2004, (47): 5798-5808, for examples of carbohydrate conjugate clusters).

As used herein, “modified carbohydrate” means any carbohydrate having one or more chemical modifications relative to naturally occurring carbohydrates.

As used herein, “carbohydrate derivative” means any compound which may be synthesized using a carbohydrate as a starting material or intermediate.

As used herein, “carbohydrate” means a naturally occurring carbohydrate, a modified carbohydrate, or a carbohydrate derivative.

As used herein “protecting group” means any compound or protecting group known to those having skill in the art. Non-limiting examples of protecting groups may be found in “Protective Groups in Organic Chemistry”, T. W. Greene, P. G. M. Wuts, ISBN 0-471-62301-6, John Wiley & Sons, Inc, New York, which is incorporated herein by reference in its entirety.

As used herein, “single-stranded” means an oligomeric compound that is not hybridized to its complement and which lacks sufficient self-complementarity to form a stable self-duplex.

As used herein, “double stranded” means a pair of oligomeric compounds that are hybridized to one another or a single self-complementary oligomeric compound that forms a hairpin structure. In certain embodiments, a double-stranded oligomeric compound comprises a first and a second oligomeric compound.

As used herein, “antisense compound” means a compound comprising or consisting of an oligonucleotide at least a portion of which is complementary to a target nucleic acid to which it is capable of hybridizing, resulting in at least one antisense activity.

As used herein, “antisense activity” means any detectable and/or measurable change attributable to the hybridization of an antisense compound to its target nucleic acid. In certain embodiments, antisense activity includes modulation of the amount or activity of a target nucleic acid transcript (e.g. mRNA). In certain embodiments, antisense activity includes modulation of the splicing of pre-mRNA.

As used herein, “RNase H based antisense compound” means an antisense compound wherein at least some of the antisense activity of the antisense compound is attributable to hybridization of the antisense compound to a target nucleic acid and subsequent cleavage of the target nucleic acid by RNase H.

As used herein, “RISC based antisense compound” means an antisense compound wherein at least some of the antisense activity of the antisense compound is attributable to the RNA Induced Silencing Complex (RISC).

As used herein, “detecting” or “measuring” means that a test or assay for detecting or measuring is performed. Such detection and/or measuring may result in a value of zero. Thus, if a test for detection or measuring results in a finding of no activity (activity of zero), the step of detecting or measuring the activity has nevertheless been performed.

As used herein, “detectable and/or measureable activity” means a statistically significant activity that is not zero.

As used herein, “essentially unchanged” means little or no change in a particular parameter, particularly relative to another parameter which changes much more. In certain embodiments, a parameter is essentially unchanged when it changes less than 5%. In certain embodiments, a parameter is essentially unchanged if it changes less than two-fold while another parameter changes at least ten-fold. For example, in certain embodiments, an antisense activity is a change in the amount of a target nucleic acid. In certain such embodiments, the amount of a non-target nucleic acid is essentially unchanged if it changes much less than the target nucleic acid does, but the change need not be zero.

As used herein, “expression” means the process by which a gene ultimately results in a protein.

Expression includes, but is not limited to, transcription, post-transcriptional modification (e.g., splicing, polyadenlyation, addition of 5′-cap), and translation.

As used herein, “target nucleic acid” means a nucleic acid molecule to which an antisense compound is intended to hybridize to result in a desired antisense activity. Antisense oligonucleotides have sufficient complementarity to their target nucleic acids to allow hybridization under physiological conditions.

As used herein, “nucleobase complementarity” or “complementarity” when in reference to nucleobases means a nucleobase that is capable of base pairing with another nucleobase. For example, in DNA, adenine (A) is complementary to thymine (T). For example, in RNA, adenine (A) is complementary to uracil (U). In certain embodiments, complementary nucleobase means a nucleobase of an antisense compound that is capable of base pairing with a nucleobase of its target nucleic acid. For example, if a nucleobase at a certain position of an antisense compound is capable of hydrogen bonding with a nucleobase at a certain position of a target nucleic acid, then the position of hydrogen bonding between the oligonucleotide and the target nucleic acid is considered to be complementary at that nucleobase pair. Nucleobases comprising certain modifications may maintain the ability to pair with a counterpart nucleobase and thus, are still capable of nucleobase complementarity.

As used herein, “non-complementary” in reference to nucleobases means a pair of nucleobases that do not form hydrogen bonds with one another.

As used herein, “complementary” in reference to oligomeric compounds (e.g., linked nucleosides, oligonucleotides, or nucleic acids) means the capacity of such oligomeric compounds or regions thereof to hybridize to another oligomeric compound or region thereof through nucleobase complementarity. Complementary oligomeric compounds need not have nucleobase complementarity at each nucleoside. Rather, some mismatches are tolerated. In certain embodiments, complementary oligomeric compounds or regions are complementary at 70% of the nucleobases (70% complementary). In certain embodiments, complementary oligomeric compounds or regions are 80% complementary. In certain embodiments, complementary oligomeric compounds or regions are 90% complementary. In certain embodiments, complementary oligomeric compounds or regions are 95% complementary. In certain embodiments, complementary oligomeric compounds or regions are 100% complementary.

As used herein, “mismatch” means a nucleobase of a first oligomeric compound that is not capable of pairing with a nucleobase at a corresponding position of a second oligomeric compound, when the first and second oligomeric compound are aligned. Either or both of the first and second oligomeric compounds may be oligonucleotides.

As used herein, “hybridization” means the pairing of complementary oligomeric compounds (e.g., an antisense compound and its target nucleic acid). While not limited to a particular mechanism, the most common mechanism of pairing involves hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleobases.

As used herein, “specifically hybridizes” means the ability of an oligomeric compound to hybridize to one nucleic acid site with greater affinity than it hybridizes to another nucleic acid site.

As used herein, “fully complementary” in reference to an oligonucleotide or portion thereof means that each nucleobase of the oligonucleotide or portion thereof is capable of pairing with a nucleobase of a complementary nucleic acid or contiguous portion thereof. Thus, a fully complementary region comprises no mismatches or unhybridized nucleobases in either strand.

As used herein, “percent complementarity” means the percentage of nucleobases of an oligomeric compound that are complementary to an equal-length portion of a target nucleic acid. Percent complementarity is calculated by dividing the number of nucleobases of the oligomeric compound that are complementary to nucleobases at corresponding positions in the target nucleic acid by the total length of the oligomeric compound.

As used herein, “percent identity” means the number of nucleobases in a first nucleic acid that are the same type (independent of chemical modification) as nucleobases at corresponding positions in a second nucleic acid, divided by the total number of nucleobases in the first nucleic acid.

As used herein, “modulation” means a change of amount or quality of a molecule, function, or activity when compared to the amount or quality of a molecule, function, or activity prior to modulation. For example, modulation includes the change, either an increase (stimulation or induction) or a decrease (inhibition or reduction) in gene expression. As a further example, modulation of expression can include a change in splice site selection of pre-mRNA processing, resulting in a change in the absolute or relative amount of a particular splice-variant compared to the amount in the absence of modulation.

As used herein, “chemical motif” means a pattern of chemical modifications in an oligonucleotide or a region thereof. Motifs may be defined by modifications at certain nucleosides and/or at certain linking groups of an oligonucleotide.

As used herein, “nucleoside motif” means a pattern of nucleoside modifications in an oligonucleotide or a region thereof. The linkages of such an oligonucleotide may be modified or unmodified. Unless otherwise indicated, motifs herein describing only nucleosides are intended to be nucleoside motifs. Thus, in such instances, the linkages are not limited.

As used herein, “sugar motif” means a pattern of sugar modifications in an oligonucleotide or a region thereof.

As used herein, “linkage motif” means a pattern of linkage modifications in an oligonucleotide or region thereof. The nucleosides of such an oligonucleotide may be modified or unmodified. Unless otherwise indicated, motifs herein describing only linkages are intended to be linkage motifs. Thus, in such instances, the nucleosides are not limited.

As used herein, “nucleobase modification motif” means a pattern of modifications to nucleobases along an oligonucleotide. Unless otherwise indicated, a nucleobase modification motif is independent of the nucleobase sequence.

As used herein, “sequence motif” means a pattern of nucleobases arranged along an oligonucleotide or portion thereof. Unless otherwise indicated, a sequence motif is independent of chemical modifications and thus may have any combination of chemical modifications, including no chemical modifications.

As used herein, “type of modification” in reference to a nucleoside or a nucleoside of a “type” means the chemical modification of a nucleoside and includes modified and unmodified nucleosides. Accordingly, unless otherwise indicated, a “nucleoside having a modification of a first type” may be an unmodified nucleoside.

As used herein, “differently modified” mean chemical modifications or chemical substituents that are different from one another, including absence of modifications. Thus, for example, a MOE nucleoside and an unmodified DNA nucleoside are “differently modified,” even though the DNA nucleoside is unmodified. Likewise, DNA and RNA are “differently modified,” even though both are naturally-occurring unmodified nucleosides. Nucleosides that are the same but for comprising different nucleobases are not differently modified. For example, a nucleoside comprising a 2′-OMe modified sugar and an unmodified adenine nucleobase and a nucleoside comprising a 2′-OMe modified sugar and an unmodified thymine nucleobase are not differently modified.

As used herein, “the same type of modifications” refers to modifications that are the same as one another, including absence of modifications. Thus, for example, two unmodified DNA nucleosides have “the same type of modification,” even though the DNA nucleoside is unmodified. Such nucleosides having the same type modification may comprise different nucleobases.

As used herein, “separate regions” means portions of an oligonucleotide wherein the chemical modifications or the motif of chemical modifications of any neighboring portions include at least one difference to allow the separate regions to be distinguished from one another.

As used herein, “pharmaceutically acceptable carrier or diluent” means any substance suitable for use in administering to an animal. In certain embodiments, a pharmaceutically acceptable carrier or diluent is sterile saline. In certain embodiments, such sterile saline is pharmaceutical grade saline.

As used herein the term “metabolic disorder” means a disease or condition principally characterized by dysregulation of metabolism—the complex set of chemical reactions associated with breakdown of food to produce energy.

As used herein, the term “Cardiovascular disease” or “cardiovascular disorder” means a disease or condition principally characterized by impaired function of the heart or blood vessels. Examples of cardiovascular diseases or disorders include, but are not limited to, aneurysm, angina, arrhythmia, atherosclerosis, cerebrovascular disease (stroke), coronary heart disease, hypertension, dyslipidemia, hyperlipidemia, and hypercholesterolemia.

As used herein the term “mono or polycyclic ring system” is meant to include all ring systems selected from single or polycyclic radical ring systems wherein the rings are fused or linked and is meant to be inclusive of single and mixed ring systems individually selected from aliphatic, alicyclic, aryl, heteroaryl, aralkyl, arylalkyl, heterocyclic, heteroaryl, heteroaromatic and heteroarylalkyl. Such mono and poly cyclic structures can contain rings that each have the same level of saturation or each, independently, have varying degrees of saturation including fully saturated, partially saturated or fully unsaturated. Each ring can comprise ring atoms selected from C, N, O and S to give rise to heterocyclic rings as well as rings comprising only C ring atoms which can be present in a mixed motif such as for example benzimidazole wherein one ring has only carbon ring atoms and the fused ring has two nitrogen atoms. The mono or polycyclic ring system can be further substituted with substituent groups such as for example phthalimide which has two ═O groups attached to one of the rings. Mono or polycyclic ring systems can be attached to parent molecules using various strategies such as directly through a ring atom, fused through multiple ring atoms, through a substituent group or through a bifunctional linking moiety.

As used herein, “prodrug” means an inactive or less active form of a compound which, when administered to a subject, is metabolized to form the active, or more active, compound (e.g., drug).

As used herein, “substituent” and “substituent group,” means an atom or group that replaces the atom or group of a named parent compound. For example a substituent of a modified nucleoside is any atom or group that differs from the atom or group found in a naturally occurring nucleoside (e.g., a modified 2′-substuent is any atom or group at the 2′-position of a nucleoside other than H or OH). Substituent groups can be protected or unprotected. In certain embodiments, compounds of the present disclosure have substituents at one or at more than one position of the parent compound. Substituents may also be further substituted with other substituent groups and may be attached directly or via a linking group such as an alkyl or hydrocarbyl group to a parent compound.

Likewise, as used herein, “substituent” in reference to a chemical functional group means an atom or group of atoms that differs from the atom or a group of atoms normally present in the named functional group.

In certain embodiments, a substituent replaces a hydrogen atom of the functional group (e.g., in certain embodiments, the substituent of a substituted methyl group is an atom or group other than hydrogen which replaces one of the hydrogen atoms of an unsubstituted methyl group). Unless otherwise indicated, groups amenable for use as substituents include without limitation, halogen, hydroxyl, alkyl, alkenyl, alkynyl, acyl (—C(O)R_(aa)), carboxyl (—C(O)O—R_(aa)), aliphatic groups, alicyclic groups, alkoxy, substituted oxy (—O—R_(aa)), aryl, aralkyl, heterocyclic radical, heteroaryl, heteroarylalkyl, amino (—N(R_(bb))(R_(cc))), imino (═NR_(bb)), amido (—C(O)N—(R_(bb))(R_(cc)) or —N(R_(bb))C(O)R_(aa)), azido (—N₃), nitro (—NO₂), cyano (—CN), carbamido (—OC(O)N(R_(bb))(R_(cc)) or —N(R_(bb))C(O)OR_(aa)), ureido (—N(R_(bb))C(O)N(R_(bb))(R_(cc))), thioureido (—N(R_(bb))C(S)N(R_(bb))(R_(cc))), guanidinyl (—N(R_(bb))C(═NR_(bb))N(R_(bb))(R_(cc))), amidinyl (—C(═NR_(bb))N(R_(bb))(R_(cc)) or —N(R_(bb))C(═NR_(bb))(R_(aa))), thiol (—SR_(bb)), sulfinyl (—S(O)R_(bb)), sulfonyl (—S(O)₂R_(bb)) and sulfonamidyl (—S(O)₂N(R_(bb))(R_(cc)) or —N(R_(bb))S(O)₂R_(bb)). Wherein each R_(aa), R_(bb) and R_(cc) is, independently, H, an optionally linked chemical functional group or a further substituent group with a preferred list including without limitation, alkyl, alkenyl, alkynyl, aliphatic, alkoxy, acyl, aryl, aralkyl, heteroaryl, alicyclic, heterocyclic and heteroarylalkyl. Selected substituents within the compounds described herein are present to a recursive degree.

As used herein, “alkyl,” as used herein, means a saturated straight or branched hydrocarbon radical containing up to twenty four carbon atoms. Examples of alkyl groups include without limitation, methyl, ethyl, propyl, butyl, isopropyl, n-hexyl, octyl, decyl, dodecyl and the like. Alkyl groups typically include from 1 to about 24 carbon atoms, more typically from 1 to about 12 carbon atoms (C₁-C₁₂ alkyl) with from 1 to about 6 carbon atoms being more preferred.

As used herein, “alkenyl,” means a straight or branched hydrocarbon chain radical containing up to twenty four carbon atoms and having at least one carbon-carbon double bond. Examples of alkenyl groups include without limitation, ethenyl, propenyl, butenyl, 1-methyl-2-buten-1-yl, dienes such as 1,3-butadiene and the like. Alkenyl groups typically include from 2 to about 24 carbon atoms, more typically from 2 to about 12 carbon atoms with from 2 to about 6 carbon atoms being more preferred. Alkenyl groups as used herein may optionally include one or more further substituent groups.

As used herein, “alkynyl,” means a straight or branched hydrocarbon radical containing up to twenty four carbon atoms and having at least one carbon-carbon triple bond. Examples of alkynyl groups include, without limitation, ethynyl, 1-propynyl, 1-butynyl, and the like. Alkynyl groups typically include from 2 to about 24 carbon atoms, more typically from 2 to about 12 carbon atoms with from 2 to about 6 carbon atoms being more preferred. Alkynyl groups as used herein may optionally include one or more further substituent groups.

As used herein, “acyl,” means a radical formed by removal of a hydroxyl group from an organic acid and has the general Formula —C(O)—X where X is typically aliphatic, alicyclic or aromatic. Examples include aliphatic carbonyls, aromatic carbonyls, aliphatic sulfonyls, aromatic sulfinyls, aliphatic sulfinyls, aromatic phosphates, aliphatic phosphates and the like. Acyl groups as used herein may optionally include further substituent groups.

As used herein, “alicyclic” means a cyclic ring system wherein the ring is aliphatic. The ring system can comprise one or more rings wherein at least one ring is aliphatic. Preferred alicyclics include rings having from about 5 to about 9 carbon atoms in the ring. Alicyclic as used herein may optionally include further substituent groups.

As used herein, “aliphatic” means a straight or branched hydrocarbon radical containing up to twenty four carbon atoms wherein the saturation between any two carbon atoms is a single, double or triple bond. An aliphatic group preferably contains from 1 to about 24 carbon atoms, more typically from 1 to about 12 carbon atoms with from 1 to about 6 carbon atoms being more preferred. The straight or branched chain of an aliphatic group may be interrupted with one or more heteroatoms that include nitrogen, oxygen, sulfur and phosphorus.

Such aliphatic groups interrupted by heteroatoms include without limitation, polyalkoxys, such as polyalkylene glycols, polyamines, and polyimines. Aliphatic groups as used herein may optionally include further substituent groups.

As used herein, “alkoxy” means a radical formed between an alkyl group and an oxygen atom wherein the oxygen atom is used to attach the alkoxy group to a parent molecule. Examples of alkoxy groups include without limitation, methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, sec-butoxy, tert-butoxy, n-pentoxy, neopentoxy, n-hexoxy and the like. Alkoxy groups as used herein may optionally include further substituent groups.

As used herein, “aminoalkyl” means an amino substituted C₁-C₁₂ alkyl radical. The alkyl portion of the radical forms a covalent bond with a parent molecule. The amino group can be located at any position and the aminoalkyl group can be substituted with a further substituent group at the alkyl and/or amino portions.

As used herein, “aralkyl” and “arylalkyl” mean an aromatic group that is covalently linked to a C₁-C₁₂ alkyl radical. The alkyl radical portion of the resulting aralkyl (or arylalkyl) group forms a covalent bond with a parent molecule. Examples include without limitation, benzyl, phenethyl and the like. Aralkyl groups as used herein may optionally include further substituent groups attached to the alkyl, the aryl or both groups that form the radical group.

As used herein, “aryl” and “aromatic” mean a mono- or polycyclic carbocyclic ring system radicals having one or more aromatic rings. Examples of aryl groups include without limitation, phenyl, naphthyl, tetrahydronaphthyl, indanyl, idenyl and the like. Preferred aryl ring systems have from about 5 to about 20 carbon atoms in one or more rings. Aryl groups as used herein may optionally include further substituent groups.

As used herein, “halo” and “halogen,” mean an atom selected from fluorine, chlorine, bromine and iodine.

As used herein, “heteroaryl,” and “heteroaromatic,” mean a radical comprising a mono- or poly-cyclic aromatic ring, ring system or fused ring system wherein at least one of the rings is aromatic and includes one or more heteroatoms. Heteroaryl is also meant to include fused ring systems including systems where one or more of the fused rings contain no heteroatoms. Heteroaryl groups typically include one ring atom selected from sulfur, nitrogen or oxygen. Examples of heteroaryl groups include without limitation, pyridinyl, pyrazinyl, pyrimidinyl, pyrrolyl, pyrazolyl, imidazolyl, thiazolyl, oxazolyl, isooxazolyl, thiadiazolyl, oxadiazolyl, thiophenyl, furanyl, quinolinyl, isoquinolinyl, benzimidazolyl, benzooxazolyl, quinoxalinyl and the like. Heteroaryl radicals can be attached to a parent molecule directly or through a linking moiety such as an aliphatic group or hetero atom. Heteroaryl groups as used herein may optionally include further substituent groups.

As used herein, “conjugate compound” means any atoms, group of atoms, or group of linked atoms suitable for use as a conjugate group. In certain embodiments, conjugate compounds may possess or impart one or more properties, including, but not limited to pharmacodynamic, pharmacokinetic, binding, absorption, cellular distribution, cellular uptake, charge and/or clearance properties.

As used herein, unless otherwise indicated or modified, the term “double-stranded” refers to two separate oligomeric compounds that are hybridized to one another. Such double stranded compounds may have one or more or non-hybridizing nucleosides at one or both ends of one or both strands (overhangs) and/or one or more internal non-hybridizing nucleosides (mismatches) provided there is sufficient complementarity to maintain hybridization under physiologically relevant conditions.

As used herein, “2′-O-methoxyethyl” (also 2′-MOE and 2′-O(CH₂)₂—OCH₃) refers to an O-methoxyethyl modification of the 2′ position ofa furosyl ring. A 2′-O-methoxyethyl modified sugar is a modified sugar.

As used herein, “2′-O-methoxyethyl nucleotide” means a nucleotide comprising a 2′-O-methoxyethyl modified sugar moiety.

“3′ target site” or “3′ stop site” refers to the nucleotide of a target nucleic acid which is complementary to the 3′-most nucleotide of a particular antisense compound.

As used herein, “5′ target site” or “5 start site” refers to the nucleotide of a target nucleic acid which is complementary to the 5′-most nucleotide of a particular antisense compound.

As used herein, “5-methylcytosine” means a cytosine modified with a methyl group attached to the 5′ position. A 5-methylcytosine is a modified nucleobase.

As used herein, “about” means within +10% of a value. For example, if it is stated, “a marker may be increased by about 50%”, it is implied that the marker may be increased between 45%-55% As used herein, “active pharmaceutical agent” means the substance or substances in a pharmaceutical composition that provide a therapeutic benefit when administered to an individual. For example, in certain embodiments an antisense oligonucleotide targeted to ANGPTL3 is an active pharmaceutical agent.

As used herein, “active target region” or “target region” means a region to which one or more active antisense compounds is targeted.

As used herein, “active antisense compounds” means antisense compounds that reduce target nucleic acid levels or protein levels.

As used herein, “adipogenesis” means the development of fat cells from preadipocytes. “Lipogenesis” means the production or formation of fat, either fatty degeneration or fatty infiltration.

As used herein, “adiposity” or “obesity” refers to the state of being obese or an excessively high amount of body fat or adipose tissue in relation to lean body mass. The amount of body fat includes concern for both the distribution of fat throughout the body and the size and mass of the adipose tissue deposits. Body fat distribution can be estimated by skin-fold measures, waist-to-hip circumference ratios, or techniques such as ultrasound, computed tomography, or magnetic resonance imaging. According to the Center for Disease Control and Prevention, individuals with a body mass index (BMI) of 30 or more are considered obese. The term “Obesity” as used herein includes conditions where there is an increase in body fat beyond the physical requirement as a result of excess accumulation of adipose tissue in the body. The term “obesity” includes, but is not limited to, the following conditions: adult-onset obesity; alimentary obesity; endogenous or metabolic obesity; endocrine obesity; familial obesity; hyperinsulinar obesity; hyperplastic-hypertrophic obesity; hypogonadal obesity; hypothyroid obesity; lifelong obesity; morbid obesity and exogenous obesity.

As used herein, “administered concomitantly” refers to the co-administration of two agents in any manner in which the pharmacological effects of both are manifest in the patient at the same time. Concomitant administration does not require that both agents be administered in a single pharmaceutical composition, in the same dosage form, or by the same route of administration. The effects of both agents need not manifest themselves at the same time. The effects need only be overlapping for a period of time and need not be coextensive.

As used herein, “administering” means providing an agent to an animal, and includes, but is not limited to, administering by a medical professional and self-administering.

As used herein, “agent” means an active substance that can provide a therapeutic benefit when administered to an animal. “First Agent” means a therapeutic compound of the invention. For example, a first agent can be an antisense oligonucleotide targeting ANGPTL3. “Second agent” means a second therapeutic compound of the invention (e.g. a second antisense oligonucleotide targeting ANGPTL3) and/or a non-ANGPTL3 therapeutic compound.

As used herein, “amelioration” refers to a lessening of at least one indicator, sign, or symptom of an associated disease, disorder, or condition. The severity of indicators can be determined by subjective or objective measures, which are known to those skilled in the art.

As used herein, “ANGPTL3” means any nucleic acid or protein of ANGPTL3.

As used herein, “ANGPTL3 expression” means the level of mRNA transcribed from the gene encoding ANGPTL3 or the level of protein translated from the mRNA. ANGPTL3 expression can be determined by art known methods such as a Northern or Western blot.

As used herein, “ANGPTL3 nucleic acid” means any nucleic acid encoding ANGPTL3. For example, in certain embodiments, an ANGPTL3 nucleic acid includes a DNA sequence encoding ANGPTL3, a RNA sequence transcribed from DNA encoding ANGPTL3 (including genomic DNA comprising introns and exons), and a mRNA sequence encoding ANGPTL3. “ANGPTL3 mRNA” means a mRNA encoding an ANGPTL3 protein.

As used herein, “animal” refers to a human or non-human animal, including, but not limited to, mice, rats, rabbits, dogs, cats, pigs, and non-human primates, including, but not limited to, monkeys and chimpanzees.

As used herein, “apoB-containing lipoprotein” means any lipoprotein that has apolipoprotein B as its protein component, and is understood to include LDL, VLDL, IDL, and lipoprotein(a) and can be generally targeted by lipid lowering agent and therapies. “ApoB-100-containing LDL” means ApoB-100 isoform containing LDL.

As used herein, “atherosclerosis” means a hardening of the arteries affecting large and medium-sized arteries and is characterized by the presence of fatty deposits. The fatty deposits are called “atheromas” or “plaques,” which consist mainly of cholesterol and other fats, calcium and scar tissue, and damage the lining of arteries.

As used herein, “cardiometabolic disease” or “cardiometabolic disorder” are diseases or disorders concerning both the cardiovascular system and the metabolic system. Examples of cardiometabolic diseases or disorders include, but are not limited to, diabetes and dyslipidemias.

As used herein, “co-administration” means administration of two or more agents to an individual.

The two or more agents can be in a single pharmaceutical composition, or can be in separate pharmaceutical compositions. Each of the two or more agents can be administered through the same or different routes of administration. Co-administration encompasses parallel or sequential administration.

As used herein, “cholesterol” is a sterol molecule found in the cell membranes of all animal tissues. Cholesterol must be transported in an animal's blood plasma by lipoproteins including very low density lipoprotein (VLDL), intermediate density lipoprotein (IDL), low density lipoprotein (LDL), and high density lipoprotein (HDL). “Plasma cholesterol” refers to the sum of all lipoproteins (VDL, IDL, LDL, HDL) esterified and/or non-estrified cholesterol present in the plasma or serum.

As used herein, “cholesterol absorption inhibitor” means an agent that inhibits the absorption of exogenous cholesterol obtained from diet.

As used herein, “coronary heart disease (CHD)” means a narrowing of the small blood vessels that supply blood and oxygen to the heart, which is often a result of atherosclerosis.

As used herein, “diabetes mellitus” or “diabetes” is a syndrome characterized by disordered metabolism and abnormally high blood sugar (hyperglycemia) resulting from insufficient levels of insulin or reduced insulin sensitivity. The characteristic symptoms are excessive urine production (polyuria) due to high blood glucose levels, excessive thirst and increased fluid intake (polydipsia) attempting to compensate for increased urination, blurred vision due to high blood glucose effects on the eye's optics, unexplained weight loss, and lethargy.

As used herein, “diabetic dyslipidemia” or “type 2 diabetes with dyslipidemia” means a condition characterized by Type 2 diabetes, reduced HDL-C, elevated triglycerides, and elevated small, dense LDL particles.

As used herein, “diluent” means an ingredient in a composition that lacks pharmacological activity, but is pharmaceutically necessary or desirable. For example, the diluent in an injected composition can be a liquid, e.g. saline solution.

As used herein, “dyslipidemia” refers to a disorder of lipid and/or lipoprotein metabolism, including lipid and/or lipoprotein overproduction or deficiency. Dyslipidemias may be manifested by elevation of lipids such as cholesterol and triglycerides as well as lipoproteins such as low-density lipoprotein (LDL) cholesterol.

As used herein, “dosage unit” means a form in which a pharmaceutical agent is provided, e.g. pill, tablet, or other dosage unit known in the art. In certain embodiments, a dosage unit is a vial containing lyophilized antisense oligonucleotide. In certain embodiments, a dosage unit is a vial containing reconstituted antisense oligonucleotide.

As used herein, “dose” means a specified quantity of a pharmaceutical agent provided in a single administration, or in a specified time period. In certain embodiments, a dose can be administered in one, two, or more boluses, tablets, or injections. For example, in certain embodiments where subcutaneous administration is desired, the desired dose requires a volume not easily accommodated by a single injection, therefore, two or more injections can be used to achieve the desired dose. In certain embodiments, the pharmaceutical agent is administered by infusion over an extended period of time or continuously. Doses can be stated as the amount of pharmaceutical agent per hour, day, week, or month. Doses can be expressed as mg/kg or g/kg.

As used herein, “effective amount” or “therapeutically effective amount” means the amount of active pharmaceutical agent sufficient to effectuate a desired physiological outcome in an individual in need of the agent. The effective amount can vary among individuals depending on the health and physical condition of the individual to be treated, the taxonomic group of the individuals to be treated, the formulation of the composition, assessment of the individual's medical condition, and other relevant factors.

As used herein, “glucose” is a monosaccharide used by cells as a source of energy and metabolic intermediate. “Plasma glucose” refers to glucose present in the plasma.

As used herein, “high density lipoprotein-C(HDL-C)” means cholesterol associated with high density lipoprotein particles. Concentration of HDL-C in serum (or plasma) is typically quantified in mg/dL or nmol/L. “serum HDL-C” and “plasma HDL-C” mean HDL-C in serum and plasma, respectively.

As used herein, “HMG-CoA reductase inhibitor” means an agent that acts through the inhibition of the enzyme HMG-CoA reductase, such as atorvastatin, rosuvastatin, fluvastatin, lovastatin, pravastatin, and simvastatin.

As used herein, “hypercholesterolemia” means a condition characterized by elevated cholesterol or circulating (plasma) cholesterol, LDL-cholesterol and VLDL-cholesterol, as per the guidelines of the Expert Panel Report of the National Cholesterol Educational Program (NCEP) of Detection, Evaluation of Treatment of high cholesterol in adults (see, Arch. Int. Med. (1988) 148, 36-39).

As used herein, “hyperlipidemia” or “hyperlipemia” is a condition characterized by elevated serum lipids or circulating (plasma) lipids. This condition manifests an abnormally high concentration of fats. The lipid fractions in the circulating blood are cholesterol, low density lipoproteins, very low density lipoproteins and triglycerides.

As used herein, “hypertriglyceridemia” means a condition characterized by elevated triglyceride levels.

As used herein, “identifying” or “selecting a subject having a metabolic or cardiovascular disease” means identifying or selecting a subject having been diagnosed with a metabolic disease, a cardiovascular disease, or a metabolic syndrome; or, identifying or selecting a subject having any symptom of a metabolic disease, cardiovascular disease, or metabolic syndrome including, but not limited to, hypercholesterolemia, hyperglycemia, hyperlipidemia, hypertriglyceridemia, hypertension, increased insulin resistance, decreased insulin sensitivity, above normal body weight, and/or above normal body fat content or any combination thereof. Such identification may be accomplished by any method, including but not limited to, standard clinical tests or assessments, such as measuring serum or circulating (plasma) cholesterol, measuring serum or circulating (plasma) blood-glucose, measuring serum or circulating (plasma) triglycerides, measuring blood-pressure, measuring body fat content, measuring body weight, and the like.

As used herein, “identifying” or “selecting a diabetic subject” means identifying or selecting a subject having been identified as diabetic or identifying or selecting a subject having any symptom of diabetes (type 1 or type 2) such as, but not limited to, having a fasting glucose of at least 110 mg/dL, glycosuria, polyuria, polydipsia, increased insulin resistance, and/or decreased insulin sensitivity.

As used herein, “identifying” or “selecting an obese subject” means identifying or selecting a subject having been diagnosed as obese or identifying or selecting a subject with a BMI over 30 and/or a waist circumference of greater than 102 cm in men or greater than 88 cm in women.

As used herein, “identifying” or “selecting a subject having dyslipidemia” means identifying or selecting a subject diagnosed with a disorder of lipid and/or lipoprotein metabolism, including lipid and/or lipoprotein overproduction or deficiency. Dyslipidemias may be manifested by elevation of lipids such as cholesterol and triglycerides as well as lipoproteins such as low-density lipoprotein (LDL) cholesterol.

As used herein, “identifying” or “selecting” a subject having increased adiposity” means identifying or selecting a subject having an increased amount of body fat (or adiposity) that includes concern for one or both the distribution of fat throughout the body and the size and mass of the adipose tissue deposits. Body fat distribution can be estimated by skin-fold measures, waist-to-hip circumference ratios, or techniques such as ultrasound, computer tomography, or magnetic resonance imaging. According to the Center for Disease Control and Prevention, individuals with a body mass index (BMI) of 30 or more are considered obese.

As used herein, “improved cardiovascular outcome” means a reduction in the occurrence of adverse cardiovascular events, or the risk thereof. Examples of adverse cardiovascular events include, without limitation, death, reinfarction, stroke, cardiogenic shock, pulmonary edema, cardiac arrest, and atrial dysrhythmia.

As used herein, “immediately adjacent” means there are no intervening elements between the immediately adjacent elements.

As used herein, “individual” or “subject” or “animal” means a human or non-human animal selected for treatment or therapy.

As used herein, “insulin resistance” is defined as the condition in which normal amounts of insulin are inadequate to produce a normal insulin response from cells, e.g., fat, muscle and/or liver cells. Insulin resistance in fat cells results in hydrolysis of stored triglycerides, which elevates free fatty acids in the blood plasma. Insulin resistance in muscle reduces glucose uptake whereas insulin resistance in liver reduces glucose storage, with both effects serving to elevate blood glucose. High plasma levels of insulin and glucose due to insulin resistance often leads to metabolic syndrome and type 2 diabetes.

As used herein, “insulin sensitivity” is a measure of how effectively an individual processes glucose.

An individual having high insulin sensitivity effectively processes glucose whereas an individual with low insulin sensitivity does not effectively process glucose.

As used herein, “intravenous administration” means administration into a vein.

As used herein, “lipid-lowering” means a reduction in one or more lipids in a subject. Lipid-lowering can occur with one or more doses over time.

As used herein, “lipid-lowering agent” means an agent, for example, an ANGPTL3-specific modulator, provided to a subject to achieve a lowering of lipids in the subject. For example, in certain embodiments, a lipid-lowering agent is provided to a subject to reduce one or more of apoB, apoC-III, total cholesterol, LDL-C, VLDL-C, IDL-C, non-HDL-C, triglycerides, small dense LDL particles, and Lp(a) in a subject.

As used herein, “lipid-lowering therapy” means a therapeutic regimen provided to a subject to reduce one or more lipids in a subject. In certain embodiments, a lipid-lowering therapy is provided to reduce one or more of apoB, apoC-III, total cholesterol, LDL-C, VLDL-C, IDL-C, non-HDL-C, triglycerides, small dense LDL particles, and Lp(a) in a subject.

As used herein, “lipoprotein”, such as VLDL, LDL and HDL, refers to a group of proteins found in the serum, plasma and lymph and are important for lipid transport. The chemical composition of each lipoprotein differs in that the HDL has a higher proportion of protein versus lipid, whereas the VLDL has a lower proportion of protein versus lipid.

As used herein, “low density lipoprotein-cholesterol (LDL-C)” means cholesterol carried in low density lipoprotein particles. Concentration of LDL-C in serum (or plasma) is typically quantified in mg/dL or nmol/L. “Serum LDL-C” and “plasma LDL-C” mean LDL-C in the serum and plasma, respectively.

As used herein, “major risk factors” refers to factors that contribute to a high risk for a particular disease or condition. In certain embodiments, major risk factors for coronary heart disease include, without limitation, cigarette smoking, hypertension, low HDL-C, family history of coronary heart disease, age, and other factors disclosed herein.

As used herein, “metabolic disorder” or “metabolic disease” refers to a condition characterized by an alteration or disturbance in metabolic function. “Metabolic” and “metabolism” are terms well known in the art and generally include the whole range of biochemical processes that occur within a living organism. Metabolic disorders include, but are not limited to, hyperglycemia, prediabetes, diabetes (type I and type 2), obesity, insulin resistance, metabolic syndrome and dyslipidemia due to type 2 diabetes.

As used herein, “metabolic syndrome” means a condition characterized by a clustering of lipid and non-lipid cardiovascular risk factors of metabolic origin. In certain embodiments, metabolic syndrome is identified by the presence of any 3 of the following factors: waist circumference of greater than 102 cm in men or greater than 88 cm in women; serum triglyceride of at least 150 mg/dL; HDL-C less than 40 mg/dL in men or less than 50 mg/dL in women; blood pressure of at least 130/85 mmHg; and fasting glucose of at least 110 mg/dL. These determinants can be readily measured in clinical practice (JAMA, 2001, 285: 2486-2497).

As used herein, “mixed dyslipidemia” means a condition characterized by elevated cholesterol and elevated triglycerides.

As used herein, “MTP inhibitor” means an agent inhibits the enzyme microsomal triglyceride transfer protein.

As used herein, “non-alcoholic fatty liver disease” or “NAFLD” means a condition characterized by fatty inflammation of the liver that is not due to excessive alcohol use (for example, alcohol consumption of over 20 g/day). In certain embodiments, NAFLD is related to insulin resistance and metabolic syndrome. NAFLD encompasses a disease spectrum ranging from simple triglyceride accumulation in hepatocytes (hepatic steatosis) to hepatic steatosis with inflammation (steatohepatitis), fibrosis, and cirrhosis.

As used herein, “nonalcoholic steatohepatitis” (NASH) occurs from progression of NAFLD beyond deposition of triglycerides. A “second hit” capable of inducing necrosis, inflammation, and fibrosis is required for development of NASH. Candidates for the second-hit can be grouped into broad categories: factors causing an increase in oxidative stress and factors promoting expression of proinflammatory cytokines. It has been suggested that increased liver triglycerides lead to increased oxidative stress in hepatocytes of animals and humans, indicating a potential cause-and-effect relationship between hepatic triglyceride accumulation, oxidative stress, and the progression of hepatic steatosis to NASH (Browning and Horton, J Clin Invest, 2004, 114, 147-152). Hypertriglyceridemia and hyperfattyacidemia can cause triglyceride accumulation in peripheral tissues (Shimamura et al., Biochem Biophys Res Commun, 2004, 322, 1080-1085).

As used herein, “nucleic acid” refers to molecules composed of monomeric nucleotides. A nucleic acid includes ribonucleic acids (RNA), deoxyribonucleic acids (DNA), single-stranded nucleic acids, double-stranded nucleic acids, small interfering ribonucleic acids (siRNA), and microRNAs (miRNA). A nucleic acid can also comprise a combination of these elements in a single molecule.

As used herein, “parenteral administration” means administration by a manner other than through the digestive tract. Parenteral administration includes topical administration, subcutaneous administration, intravenous administration, intramuscular administration, intraarterial administration, intraperitoneal administration, or intracranial administration, e.g. intrathecal or intracerebroventricular administration. Administration can be continuous, or chronic, or short or intermittent.

As used herein, “pharmaceutical agent” means a substance that provides a therapeutic benefit when administered to an individual. For example, in certain embodiments, an antisense oligonucleotide targeted to ANGPTL3 is pharmaceutical agent.

As used herein, “pharmaceutical composition” means a mixture of substances suitable for administering to an individual. For example, a pharmaceutical composition can comprise one or more active agents and a sterile aqueous solution.

As used herein, “pharmaceutically acceptable carrier” means a medium or diluent that does not interfere with the structure or function of the oligonucleotide. Certain, of such carries enable pharmaceutical compositions to be formulated as, for example, tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspension and lozenges for the oral ingestion by a subject. Certain of such carriers enable pharmaceutical compositions to be formulated for injection or infusion. For example, a pharmaceutically acceptable carrier can be a sterile aqueous solution.

As used herein, “pharmaceutically acceptable salts” means physiologically and pharmaceutically acceptable salts of antisense compounds, i.e., salts that retain the desired biological activity of the parent oligonucleotide and do not impart undesired toxicological effects thereto.

As used herein, “portion” means a defined number of contiguous (i.e. linked) nucleobases of a nucleic acid. In certain embodiments, a portion is a defined number of contiguous nucleobases of a target nucleic acid. In certain embodiments, a portion is a defined number of contiguous nucleobases of an antisense compound.

As used herein, “prevent” refers to delaying or forestalling the onset or development of a disease, disorder, or condition for a period of time from minutes to indefinitely. Prevent also means reducing risk of developing a disease, disorder, or condition.

As used herein, “side effects” means physiological responses attributable to a treatment other than the desired effects. In certain embodiments, side effects include injection site reactions, liver function test abnormalities, renal function abnormalities, liver toxicity, renal toxicity, central nervous system abnormalities, myopathies, and malaise. For example, increased aminotransferase levels in serum can indicate liver toxicity or liver function abnormality. For example, increased bilirubin can indicate liver toxicity or liver function abnormality.

As used herein, “statin” means an agent that inhibits the activity of HMG-CoA reductase.

As used herein, “subcutaneous administration” means administration just below the skin.

As used herein, “targeting” or “targeted” means the process of design and selection of an antisense compound that will specifically hybridize to a target nucleic acid and induce a desired effect.

As used herein, “target nucleic acid,” “target RNA,” and “target RNA transcript” all refer to a nucleic acid capable of being targeted by antisense compounds.

As used herein, “target region” is defined as a portion of the target nucleic acid having at least one identifiable structure, function, or characteristic.

As used herein, “target segment” means the sequence of nucleotides of a target nucleic acid to which one or more antisense compound is targeted. “5′ target site” or “5′ start site” refers to the 5′-most nucleotide of a target segment. “3′ target site” or “3′ stop site” refers to the 3′-most nucleotide of a target segment.

As used herein, “therapeutically effective amount” means an amount of an agent that provides a therapeutic benefit to an individual.

As used herein, “therapeutic lifestyle change” means dietary and lifestyle changes intended to lower fat/adipose tissue mass and/or cholesterol. Such change can reduce the risk of developing heart disease, and may include recommendations for dietary intake of total daily calories, total fat, saturated fat, polyunsaturated fat, monounsaturated fat, carbohydrate, protein, cholesterol, insoluble fiber, as well as recommendations for physical activity.

As used herein, “triglyceride” means a lipid or neutral fat consisting of glycerol combined with three fatty acid molecules.

As used herein, “type 2 diabetes” (also known as “type 2 diabetes mellitus” or “diabetes mellitus, type 2”, and formerly called “diabetes mellitus type 2”, “non-insulin-dependent diabetes (NIDDM)”, “obesity related diabetes”, or “adult-onset diabetes”) is a metabolic disorder that is primarily characterized by insulin resistance, relative insulin deficiency, and hyperglycemia.

As used herein, “treat” refers to administering a pharmaceutical composition to effect an alteration or improvement of a disease, disorder, or condition.

Certain Embodiments

In certain embodiments disclosed herein, ANGPTL3 has the sequence as set forth in GenBank Accession No. NM_014495.2 (incorporated herein as SEQ ID NO: 1). In certain embodiments, ANGPTL3 has the sequence as set forth in GenBank Accession No. NT_032977.9 nucleotides 33032001 to 33046000 (incorporated herein as SEQ ID NO: 2).

Certain embodiments disclosed herein provide compounds or compositions comprising a modified oligonucleotide and a conjugate group, wherein the modified oligonucleotide consists of 12 to 30 nucleosides having a nucleobase sequence comprising at least 8 contiguous nucleobases complementary to an equal length portion of SEQ ID NOs: 1-2.

In certain embodiments, a compound comprises a siRNA or antisense oligonucleotide targeted to ANGPTL3 known in the art and a conjugate group described herein. Examples of antisense oligonucleotides targeted to ANGPTL3 suitable for conjugation include but are not limited to those disclosed in U.S. Pat. No. 8,653,047 (WO 2011/085271), which is incorporated by reference in its entirety herein. In certain embodiments, a compound comprises an antisense oligonucleotide having a nucleobase sequence of any of SEQ ID NOs: 34-111 disclosed in U.S. Pat. No. 8,653,047 and a conjugate group described herein. In certain embodiments, a compound comprises a siRNA sense or antisense strand having a nucleobase sequence of any of SEQ ID NOs: 34-111 disclosed in U.S. Pat. No. 8,653,047 and a conjugate group described herein. The nucleobase sequences of all of the aforementioned referenced SEQ ID NOs are incorporated by reference herein.

Certain embodiments disclosed herein provide compounds or compositions comprising a modified oligonucleotide and a conjugate group, wherein the modified oligonucleotide consists of 12 to 30 linked nucleosides in length targeted to ANGPTL3. The ANGPTL3 target can have a sequence selected from any one of SEQ ID NOs: 1-2.

Certain embodiments disclosed herein provide compounds or compositions comprising a modified oligonucleotide and a conjugate group, wherein the modified oligonucleotide consists of 12 to 30 linked nucleosides and comprising a nucleobase sequence comprising a portion of at least 8 contiguous nucleobases complementary to an equal length portion of nucleobases 1140 to 1159 of SEQ ID NO: 1, wherein the nucleobase sequence of the modified oligonucleotide is at least 80% complementary to SEQ ID NO: 1. In certain embodiments, the modified oligonucleotide is at least 8, least 9, least 10, least 11, at least 12, least 13, at least 14, at least 15, at least 16, least 17, least 18, least 19, or 20 contiguous nucleobases complementary to an equal length portion of nucleobases 1140 to 1159 of SEQ ID NO: 1.

Certain embodiments disclosed herein provide compounds or compositions comprising a modified oligonucleotide and a conjugate group, wherein the modified oligonucleotide consists of 12 to 30 linked nucleosides and comprising a nucleobase sequence complementary to nucleobases 1140 to 1159 of SEQ ID NO: 1, wherein the nucleobase sequence of the modified oligonucleotide is at least 80% complementary to SEQ ID NO: 1.

Certain embodiments disclosed herein provide compounds or compositions comprising a modified oligonucleotide and a conjugate group, wherein the modified oligonucleotide consists of 12 to 30 linked nucleosides and comprising a nucleobase sequence comprising a portion of at least 8 contiguous nucleobases complementary to an equal length portion of nucleobases 1907 to 1926 of SEQ ID NO: 1, wherein the nucleobase sequence of the modified oligonucleotide is at least 80% complementary to SEQ ID NO: 1. In certain embodiments, the modified oligonucleotide is at least 8, least 9, least 10, least 11, at least 12, least 13, at least 14, at least 15, at least 16, least 17, least 18, least 19, or 20 contiguous nucleobases complementary to an equal length portion of nucleobases 1907 to 1926 of SEQ ID NO: 1.

Certain embodiments disclosed herein provide compounds or compositions comprising a modified oligonucleotide and a conjugate group, wherein the modified oligonucleotide consists of 12 to 30 linked nucleosides and comprising a nucleobase sequence complementary to nucleobases 1907 to 1926 of SEQ ID NO: 1, wherein the nucleobase sequence of the modified oligonucleotide is at least 80% complementary to SEQ ID NO: 1.

Certain embodiments disclosed herein provide compounds or compositions comprising a modified oligonucleotide and a conjugate group, wherein the modified oligonucleotide consists of 12 to 30 linked nucleosides and comprising a nucleobase sequence comprising a portion of at least 8 contiguous nucleobases complementary to an equal length portion of nucleobases 147 to 162 of SEQ ID NO: 1, wherein the nucleobase sequence of the modified oligonucleotide is at least 80% complementary to SEQ ID NO: 1. In certain embodiments, the modified oligonucleotide is at least 8, least 9, least 10, least 11, at least 12, least 13, at least 14, at least 15, or 16 contiguous nucleobases complementary to an equal length portion of nucleobases 147 to 162 of SEQ ID NO: 1.

Certain embodiments disclosed herein provide compounds or compositions comprising a modified oligonucleotide and a conjugate group, wherein the modified oligonucleotide consists of 12 to 30 linked nucleosides and comprising a nucleobase sequence complementary to nucleobases 147 to 162 of SEQ ID NO: 1, wherein the nucleobase sequence of the modified oligonucleotide is at least 80% complementary to SEQ ID NO: 1.

In certain embodiments, the modified oligonucleotide consists of 12 to 30, 15 to 30, 18 to 24, 19 to 22, 13 to 25, 14 to 25, 15 to 25 or 16 to 24 linked nucleosides. In certain embodiments, the modified oligonucleotide consists of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 linked nucleosides or a range defined by any two of these values. In certain embodiments, the modified oligonucleotide is 16 linked nucleosides in length. In certain embodiments, the modified oligonucleotide is 20 linked nucleosides in length.

In certain embodiments, the modified oligonucleotide comprises a nucleobase sequence comprising a portion of at least 8, least 9, least 10, least 11, at least 12, least 13, at least 14, at least 15, at least 16, least 17, least 18, least 19, or 20 contiguous nucleobases complementary to an equal length portion of SEQ ID NO: 1 or 2.

Certain embodiments disclosed herein provide compounds or compositions comprising a modified oligonucleotide and a conjugate group, wherein the modified oligonucleotide consists of 12 to 30 linked nucleosides and having a nucleobase sequence comprising at least 8, least 9, least 10, least 11, at least 12, least 13, at least 14, at least 15, at least 16, least 17, least 18, least 19, or 20 contiguous nucleobases of a nucleobase sequence selected from any one of SEQ ID NOs: 15-27, 30-73, 75-85, 87-232, 238, 240-243, 245-247, 249-262, 264-397, 399-469, 471-541, 543-600, 604-760, 762-819, 821-966, 968-971, 973-975, 977-990, 992-1110, 1112-1186, 1188-1216, 1218-1226, 1228-1279, 1281-1293, 1295-1304, 1306-1943, 1945-1951, 1953-1977, 1979-1981, 1983-2044, 2046-2097, 2099-2181, 2183-2232, 2234-2238, 2240-2258, 2260-2265, 2267-2971, 2973-2976, 2978-4162, 4164-4329, 4331-4389, 4391-4394, 4396-4877.

Certain embodiments disclosed herein provide compounds or compositions comprising a modified oligonucleotide and a conjugate group, wherein the modified oligonucleotide consists of 12 to 30 linked nucleosides and having a nucleobase sequence comprising at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or 20 contiguous nucleobases of the nucleobase sequences of SEQ ID NO: 77. In certain embodiments, the compound comprises ISIS 563580 and a conjugate group. In certain embodiments, the compound consists of ISIS 563580 and a conjugate group.

In certain embodiments, the present disclosure provides conjugated antisense compounds represented by the following structure. In certain embodiments, the antisense compound comprises the modified oligonucleotide ISIS 563580 with a 5′-X, wherein X is a conjugate group comprising GalNAc. In certain embodiments, the antisense compound consists of the modified oligonucleotide ISIS 563580 with a 5′-X, wherein X is a conjugate group comprising GalNAc.

In certain embodiments, the present disclosure provides conjugated antisense compounds represented by the following structure. In certain embodiments, the antisense compound comprises the conjugated modified oligonucleotide ISIS 703801. In certain embodiments, the antisense compound consists of the conjugated modified oligonucleotide ISIS 703801.

In certain embodiments, the present disclosure provides conjugated antisense compounds represented by the following structure. In certain embodiments, the antisense compound comprises the conjugated modified oligonucleotide ISIS 703802. In certain embodiments, the antisense compound consists of the conjugated modified oligonucleotide ISIS 703802.

In certain embodiments, the present disclosure provides conjugated antisense compounds represented by the following structure. In certain embodiments, the antisense compound comprises a modified oligonucleotide with the nucleobase sequence of SEQ ID NO: 77 with a 5′-GalNAc with variability in the sugar mods of the wings. In certain embodiments, the antisense compound consists of a modified oligonucleotide with the nucleobase sequence of SEQ ID NO: 77 with a 5′-GalNAc with variability in the sugar mods of the wings.

wherein either R¹ is —OCH₂CH₂OCH₃ (MOE) and R² is H; or R¹ and R² together form a bridge, wherein R¹ is —O— and R² is —CH₂—, —CH(CH₃)—, or —CH₂CH₂—, and R¹ and R² are directly connected such that the resulting bridge is selected from: —O—CH₂—, —O—CH(CH₃)—, and —O—CH₂CH₂—;

and for each pair of R³ and R⁴ on the same ring, independently for each ring: either R³ is selected from H and —OCH₂CH₂OCH₃ and R⁴ is H; or R³ and R⁴ together form a bridge, wherein R³ is —O—, and R⁴ is —CH₂—, —CH(CH₃)—, or —CH₂CH₂— and R³ and R⁴ are directly connected such that the resulting bridge is selected from: —O—CH₂—, —O—CH(CH₃)—, and —O—CH₂CH₂—;

and R⁵ is selected from H and —CH₃;

and Z is selected from S— and O—.

Certain embodiments disclosed herein provide compounds or compositions comprising a modified oligonucleotide and a conjugate group, wherein the modified oligonucleotide consists of 12 to 30 linked nucleosides and having a nucleobase sequence comprising at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or 20 contiguous nucleobases of the nucleobase sequence of SEQ ID NO: 20. In certain embodiments, the compound comprises ISIS 544199 and a conjugate group. In certain embodiments, the compound consists of ISIS 544199 and a conjugate group.

Certain embodiments disclosed herein provide compounds or compositions comprising a modified oligonucleotide and a conjugate group, wherein the modified oligonucleotide consists of 12 to 30 linked nucleosides and having a nucleobase sequence comprising at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or 20 contiguous nucleobases of the nucleobase sequence of SEQ ID NO: 35. In certain embodiments, the compound comprises ISIS 560400 and a conjugate group. In certain embodiments, the compound consists of ISIS 560400 and a conjugate group.

Certain embodiments disclosed herein provide compounds or compositions comprising a modified oligonucleotide and a conjugate group, wherein the modified oligonucleotide consists of 12 to 30 linked nucleosides and having a nucleobase sequence comprising at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or 20 contiguous nucleobases of the nucleobase sequence of SEQ ID NO: 90. In certain embodiments, the compound comprises ISIS 567233 and a conjugate group. In certain embodiments, the compound consists of ISIS 567233 and a conjugate group.

Certain embodiments disclosed herein provide compounds or compositions comprising a modified oligonucleotide and a conjugate group, wherein the modified oligonucleotide consists of 12 to 30 linked nucleosides and having a nucleobase sequence comprising at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or 20 contiguous nucleobases of the nucleobase sequence of SEQ ID NO: 93. In certain embodiments, the compound comprises ISIS 567320 and a conjugate group. In certain embodiments, the compound consists of ISIS 567320 and a conjugate group.

Certain embodiments disclosed herein provide compounds or compositions comprising a modified oligonucleotide and a conjugate group, wherein the modified oligonucleotide consists of 12 to 30 linked nucleosides and having a nucleobase sequence comprising at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or 20 contiguous nucleobases of the nucleobase sequence of SEQ ID NO: 94. In certain embodiments, the compound comprises ISIS 567321 and a conjugate group. In certain embodiments, the compound consists of ISIS 567321 and a conjugate group.

Certain embodiments disclosed herein provide compounds or compositions comprising a modified oligonucleotide and a conjugate group, wherein the modified oligonucleotide consists of 12 to 30 linked nucleosides and having a nucleobase sequence comprising at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, or 16 contiguous nucleobases of the nucleobase sequence of SEQ ID NO: 110. In certain embodiments, the compound comprises ISIS 559277 and a conjugate group. In certain embodiments, the compound consists of ISIS 559277 and a conjugate group.

Certain embodiments disclosed herein provide compounds or compositions comprising a modified oligonucleotide and a conjugate group, wherein the modified oligonucleotide consists of 12 to 30 linked nucleosides and having a nucleobase sequence comprising at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, or 16 contiguous nucleobases of the nucleobase sequence of SEQ ID NO: 114. In certain embodiments, the compound comprises ISIS 561011 and a conjugate group. In certain embodiments, the compound consists of ISIS 561011 and a conjugate group.

In certain embodiments, the nucleobase sequence of the modified oligonucleotide is at least 70%, 75%, 80%, 85%, 90%, 95% or 100% complementary to any one of SEQ ID NO: 1-2 as measured over the entirety of the modified oligonucleotide.

In certain embodiments, the compound disclosed herein is a single-stranded oligonucleotide. In certain embodiments, the compound disclosed herein is a single-stranded modified oligonucleotide.

In certain embodiments, at least one internucleoside linkage of said modified oligonucleotide is a modified internucleoside linkage. In certain embodiments, the modified internucleoside linkage is a phosphorothioate internucleoside linkage. In certain embodiments, at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9 or at least 10 internucleoside linkages of said modified oligonucleotide are phosphorothioate internucleoside linkages. In certain embodiments, each internucleoside linkage is a phosphorothioate internucleoside linkage. In certain embodiments, the modified oligonucleotide comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9 or at least 10 phosphodiester internucleoside linkages. In certain embodiments, each internucleoside linkage of the modified oligonucleotide is selected from a phosphodiester internucleoside linkage and a phosphorothioate internucleoside linkage.

In certain embodiments, at least one nucleoside of the modified oligonucleotide comprises a modified sugar. In certain embodiments, at least one modified sugar is a bicyclic sugar. In certain embodiments, at least one modified sugar comprises a 2′-O-methoxyethyl, a constrained ethyl, a 3′-fluoro-HNA or a 4′-(CH₂)_(n)—O-2′ bridge, wherein n is 1 or 2.

In certain embodiments, at least one nucleoside of said modified oligonucleotide comprises a modified nucleobase. In certain embodiments, the modified nucleobase is a 5-methylcytosine.

Certain embodiments disclosed herein provide compounds or compositions comprising a modified oligonucleotide and a conjugate group, wherein the modified oligonucleotide has: a) a gap segment consisting of linked deoxynucleosides; b) a 5′ wing segment consisting of linked nucleosides; and c) a 3′ wing segment consisting of linked nucleosides. The gap segment is positioned between the 5′ wing segment and the 3′ wing segment and each nucleoside of each wing segment comprises a modified sugar.

In certain embodiments, the modified oligonucleotide consists of 12 to 30 linked nucleosides and comprises: a gap segment consisting of linked deoxynucleosides; a 5′ wing segment consisting of linked nucleosides; a 3′ wing segment consisting of linked nucleosides; wherein the gap segment is positioned between the 5′ wing segment and the 3′ wing segment and wherein each nucleoside of each wing segment comprises a modified sugar.

In certain embodiments, the compounds or compositions disclosed herein comprise a modified oligonucleotide consisting of 20 linked nucleosides having a nucleobase sequence comprising at least 8 contiguous nucleobases complementary to an equal length portion of SEQ ID NO: 1-2, wherein the modified oligonucleotide comprises: a gap segment consisting of ten linked deoxynucleosides; a 5′ wing segment consisting of five linked nucleosides; and a 3′ wing segment consisting of five linked nucleosides; wherein the gap segment is positioned between the 5′ wing segment and the 3′ wing segment; wherein each nucleoside of each wing segment comprises a 2′-O-methoxyethyl sugar; wherein at least one internucleoside linkage is a phosphorothioate linkage and wherein each cytosine residue is a 5-methylcytosine. In certain embodiments, each internucleoside linkage is a phosphorothioate linkage.

In certain embodiments, the modified oligonucleotide consists of 20 linked nucleosides and comprises: a gap segment consisting of ten linked deoxynucleosides; a 5′ wing segment consisting of five linked nucleosides; a 3′ wing segment consisting of five linked nucleosides; wherein the gap segment is positioned between the 5′ wing segment and the 3′ wing segment; wherein each nucleoside of each wing segment comprises a 2′-O-methoxyethyl sugar; wherein at least one internucleoside linkage is a phosphorothioate linkage and wherein each cytosine residue is a 5-methylcytosine. In certain embodiments, each internucleoside linkage is a phosphorothioate linkage.

In certain embodiments, the compounds or compositions disclosed herein comprise a modified oligonucleotide consisting of 20 linked nucleosides having a nucleobase sequence comprising at least 8 contiguous nucleobases of a nucleobase sequence selected of SEQ ID NO: 77, wherein the modified oligonucleotide comprises: a gap segment consisting of ten linked deoxynucleosides; a 5′ wing segment consisting of five linked nucleosides; and a 3′ wing segment consisting of five linked nucleosides; wherein the gap segment is positioned between the 5′ wing segment and the 3′ wing segment; wherein each nucleoside of each wing segment comprises a 2′-O-methoxyethyl sugar; wherein at least one internucleoside linkage is a phosphorothioate linkage and wherein each cytosine residue is a 5-methylcytosine. In certain embodiments, each internucleoside linkage is a phosphorothioate linkage.

In certain embodiments, the modified oligonucleotide consists of 20 linked nucleosides with the nucleobase sequence of SEQ ID NO: 77 and comprises: a gap segment consisting often linked deoxynucleosides; a 5′ wing segment consisting of five linked nucleosides; a 3′ wing segment consisting of five linked nucleosides; wherein the gap segment is positioned between the 5′ wing segment and the 3′ wing segment; wherein each nucleoside of each wing segment comprises a 2′-O-methoxyethyl sugar; wherein at least one internucleoside linkage is a phosphorothioate linkage and wherein each cytosine residue is a 5-methylcytosine. In certain embodiments, each internucleoside linkage is a phosphorothioate linkage.

In certain embodiments, the compounds or compositions disclosed herein comprise a modified oligonucleotide consisting of 20 linked nucleosides having a nucleobase sequence comprising at least 8 contiguous nucleobases of a nucleobase sequence selected of SEQ ID NO: 20, wherein the modified oligonucleotide comprises: a gap segment consisting of ten linked deoxynucleosides; a 5′ wing segment consisting of five linked nucleosides; and a 3′ wing segment consisting of five linked nucleosides; wherein the gap segment is positioned between the 5′ wing segment and the 3′ wing segment; wherein each nucleoside of each wing segment comprises a 2′-O-methoxyethyl sugar; wherein at least one internucleoside linkage is a phosphorothioate linkage and wherein each cytosine residue is a 5-methylcytosine. In certain embodiments, each internucleoside linkage is a phosphorothioate linkage.

In certain embodiments, the modified oligonucleotide consists of 20 linked nucleosides with the nucleobase sequence of SEQ ID NO: 20 and comprises: a gap segment consisting often linked deoxynucleosides; a 5′ wing segment consisting of five linked nucleosides; a 3′ wing segment consisting of five linked nucleosides; wherein the gap segment is positioned between the 5′ wing segment and the 3′ wing segment; wherein each nucleoside of each wing segment comprises a 2′-O-methoxyethyl sugar; wherein at least one internucleoside linkage is a phosphorothioate linkage and wherein each cytosine residue is a 5-methylcytosine. In certain embodiments, each internucleoside linkage is a phosphorothioate linkage.

In certain embodiments, the compounds or compositions disclosed herein comprise a modified oligonucleotide consisting of 16 linked nucleosides having a nucleobase sequence comprising at least 8 contiguous nucleobases of a nucleobase sequence of SEQ ID NO: 110, wherein the modified oligonucleotide comprises: a gap segment consisting of ten linked deoxynucleosides; a 5′ wing segment consisting of three linked nucleosides; and a 3′ wing segment consisting of three linked nucleosides; wherein the gap segment is positioned between the 5′ wing segment and the 3′ wing segment; wherein each wing segment comprises at least one 2′-O-methoxyethyl sugar and at least one cEt sugar; wherein at least one internucleoside linkage is a phosphorothioate linkage and wherein each cytosine residue is a 5-methylcytosine. In certain embodiments, each internucleoside linkage is a phosphorothioate linkage.

In certain embodiments, the modified oligonucleotide consists of 16 linked nucleosides with the nucleobase sequence of SEQ ID NO: 110 and comprises: a gap segment consisting of ten linked deoxynucleosides; a 5′ wing segment consisting of three linked nucleosides; a 3′ wing segment consisting of three linked nucleosides; wherein the gap segment is positioned between the 5′ wing segment and the 3′ wing segment; wherein each wing segment comprises at least one 2′-O-methoxyethyl sugar and at least one cEt sugar; wherein at least one internucleoside linkage is a phosphorothioate linkage and wherein each cytosine residue is a 5-methylcytosine. In certain embodiments, each internucleoside linkage is a phosphorothioate linkage.

In certain embodiments, the conjugate group is linked to the modified oligonucleotide at the 5′ end of the modified oligonucleotide. In certain embodiments, the conjugate group is linked to the modified oligonucleotide at the 3′ end of the modified oligonucleotide.

In certain embodiments, the conjugate group comprises exactly one ligand. In certain embodiments, the conjugate group comprises one or more ligands. In certain embodiments, the conjugate group comprises exactly two ligands. In certain embodiments, the conjugate group comprises two or more ligands. In certain embodiments, the conjugate group comprises three or more ligands. In certain embodiments, the conjugate group comprises exactly three ligands. In certain embodiments, each ligand is selected from among: a polysaccharide, modified polysaccharide, mannose, galactose, a mannose derivative, a galactose derivative, D-mannopyranose, L-Mannopyranose, D-Arabinose, L-Galactose, D-xylofuranose, L-xylofuranose, D-glucose, L-glucose, D-Galactose, L-Galactose, α-D-Mannofuranose, β-D-Mannofuranose, α-D-Mannopyranose, β-D-Mannopyranose, α-D-Glucopyranose, β-D-Glucopyranose, α-D-Glucofuranose, β-D-Glucofuranose, α-D-fructofuranose, α-D-fructopyranose, α-D-Galactopyranose, β-D-Galactopyranose, α-D-Galactofuranose, β-D-Galactofuranose, glucosamine, sialic acid, α-D-galactosamine, N-Acetylgalactosamine, 2-Amino-3-O—[(R)-1-carboxyethyl]-2-deoxy-β-D-glucopyranose, 2-Deoxy-2-methylamino-L-glucopyranose, 4,6-Dideoxy-4-formamido-2,3-di-O-methyl-D-mannopyranose, 2-Deoxy-2-sulfoamino-D-glucopyranose, N-Glycoloyl-α-neuraminic acid, 5-thio-β-D-glucopyranose, methyl 2,3,4-tri-O-acetyl-1-thio-6-O-trityl-α-D-glucopyranoside, 4-Thio-β-D-galactopyranose, ethyl 3,4,6,7-tetra-O-acetyl-2-deoxy-1,5-dithio-α-D-gluco-heptopyranoside, 2,5-Anhydro-D-allononitrile, ribose, D-ribose, D-4-thioribose, L-ribose, L-4-thioribose. In certain embodiments, each ligand is N-acetyl galactosamine.

In certain embodiments, the conjugate group comprises:

In certain embodiments, the conjugate group comprises:

In certain embodiments, the conjugate group comprises:

In certain embodiments, the conjugate group comprises:

In certain embodiments, the conjugate group comprises:

In certain embodiments, the conjugate group comprises at least one phosphorus linking group or neutral linking group.

In certain embodiments, the conjugate group comprises a structure selected from among:

-   -   wherein n is from 1 to 12; and     -   wherein m is from 1 to 12.

In certain embodiments, the conjugate group has a tether having a structure selected from among:

wherein L is either a phosphorus linking group or a neutral linking group;

-   -   Z₁ is C(═O)O—R₂;     -   Z₂ is H, C₁-C₆ alkyl or substituted C₁-C₆ alky;     -   R₂ is H, C₁-C₆ alkyl or substituted C₁-C₆ alky; and     -   each m₁ is, independently, from 0 to 20 wherein at least one m₁         is greater than 0 for each tether.

In certain embodiments, the conjugate group has a tether having a structure selected from among:

-   -   wherein Z₂ is H or CH₃; and     -   each m₁ is, independently, from 0 to 20 wherein at least one m₁         is greater than 0 for each tether.

In certain embodiments, the conjugate group has tether having a structure selected from among:

-   -   wherein n is from 1 to 12; and     -   wherein m is from 1 to 12.

In certain embodiments, the conjugate group is covalently attached to the modified oligonucleotide.

In certain embodiments, the compound has a structure represented by the formula: A-B—C-D

E-F)_(q)

-   -   wherein     -   A is the modified oligonucleotide;     -   B is the cleavable moiety     -   C is the conjugate linker     -   D is the branching group     -   each E is a tether;     -   each F is a ligand; and     -   q is an integer between 1 and 5.

In certain embodiments, the compound has a structure represented by the formula: A

B

_(n) ₂

C

_(n) ₁

D

_(n) ₃

E-F

_(q)

-   -   wherein:     -   A is the modified oligonucleotide;     -   B is the cleavable moiety     -   C is the conjugate linker     -   D is the branching group     -   each E is a tether;     -   each F is a ligand;     -   each n is independently 0 or 1; and     -   q is an integer between 1 and 5.

In certain embodiments, the compound has a structure represented by the formula: A-B—C

E-F)_(q)

-   -   wherein     -   A is the modified oligonucleotide;     -   B is the cleavable moiety;     -   C is the conjugate linker;     -   each E is a tether;     -   each F is a ligand; and     -   q is an integer between 1 and 5.

In certain embodiments, the compound has a structure represented by the formula: A-C-D-

E-F)_(q)

-   -   wherein     -   A is the modified oligonucleotide;     -   C is the conjugate linker;     -   D is the branching group;     -   each E is a tether;     -   each F is a ligand; and     -   q is an integer between 1 and 5.

In certain embodiments, the compound has a structure represented by the formula: A-C-

E-F)_(q)

-   -   wherein     -   A is the modified oligonucleotide;     -   C is the conjugate linker;     -   each E is a tether;     -   each F is a ligand; and     -   q is an integer between 1 and 5.

In certain embodiments, the compound has a structure represented by the formula: A-B-D

E-F)_(q)

-   -   wherein     -   A is the modified oligonucleotide;     -   B is the cleavable moiety;     -   D is the branching group;     -   each E is a tether;     -   each F is a ligand; and     -   q is an integer between 1 and 5.

In certain embodiments, the compound has a structure represented by the formula: A-B-D

E-F)_(q)

-   -   wherein     -   A is the modified oligonucleotide;     -   B is the cleavable moiety;     -   each E is a tether;     -   each F is a ligand; and     -   q is an integer between 1 and 5.

In certain embodiments, the compound has a structure represented by the formula: A-D

E-F)_(q)

-   -   wherein     -   A is the modified oligonucleotide;     -   D is the branching group;     -   each E is a tether;     -   each F is a ligand; and     -   q is an integer between 1 and 5.

In certain embodiments, the conjugate linker has a structure selected from among:

wherein each L is, independently, a phosphorus linking group or a neutral linking group; and

each n is, independently, from 1 to 20.

In certain embodiments, the conjugate linker has a structure selected from among:

In certain embodiments, the conjugate linker has the following structure:

In certain embodiments, the conjugate linker has a structure selected from among:

In certain embodiments, the conjugate linker has a structure selected from among:

In certain embodiments, the conjugate linker has a structure selected from among:

In certain embodiments, the conjugate linker comprises a pyrrolidine. In certain embodiments, the conjugate linker does not comprise a pyrrolidine.

In certain embodiments, the conjugate linker comprises PEG.

In certain embodiments, the conjugate linker comprises an amide. In certain embodiments, the conjugate linker comprises at least two amides. In certain embodiments, the conjugate linker does not comprise an amide. In certain embodiments, the conjugate linker comprises a polyamide.

In certain embodiments, the conjugate linker comprises an amine.

In certain embodiments, the conjugate linker comprises one or more disulfide bonds.

In certain embodiments, the conjugate linker comprises a protein binding moiety. In certain embodiments, the protein binding moiety comprises a lipid. In certain embodiments, the protein binding moiety is selected from among: cholesterol, cholic acid, adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone, 1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine), a vitamin (e.g., folate, vitamin A, vitamin E, biotin, pyridoxal), a peptide, a carbohydrate (e.g., monosaccharide, disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, polysaccharide), an endosomolytic component, a steroid (e.g., uvaol, hecigenin, diosgenin), a terpene (e.g., triterpene, e.g., sarsasapogenin, friedelin, epifriedelanol derivatized lithocholic acid), or a cationic lipid. In certain embodiments, the protein binding moiety is selected from among: a C16 to C22 long chain saturated or unsaturated fatty acid, cholesterol, cholic acid, vitamin E, adamantane or 1-pentafluoropropyl.

In certain embodiments, the conjugate linker has a structure selected from among:

wherein each n is, independently, is from 1 to 20; and p is from 1 to 6.

In certain embodiments, the conjugate linker has a structure selected from among:

wherein each n is, independently, from 1 to 20.

In certain embodiments, the conjugate linker has a structure selected from among:

In certain embodiments, the conjugate linker has a structure selected from among:

wherein n is from 1 to 20.

In certain embodiments, the conjugate linker has a structure selected from among:

In certain embodiments, the conjugate linker has a structure selected from among:

wherein each n is independently, 0, 1, 2, 3, 4, 5, 6, or 7.

In certain embodiments, the conjugate linker has the following structure:

In certain embodiments, the branching group has one of the following structures:

wherein each A₁ is independently, O, S, C═O or NH; and

each n is, independently, from 1 to 20.

In certain embodiments, the branching group has one of the following structures:

-   -   wherein each A₁ is independently, O, S, C═O or NH; and     -   each n is, independently, from 1 to 20.

In certain embodiments, the branching group has the following structure:

In certain embodiments, the branching group has the following structure:

In certain embodiments, the branching group has the following structure:

In certain embodiments, the branching group has the following structure:

In certain embodiments, the branching group comprises an ether.

In certain embodiments, the branching group has the following structure:

each n is, independently, from 1 to 20; and

m is from 2 to 6.

In certain embodiments, the branching group has the following structure:

In certain embodiments, the branching group has the following structure:

In certain embodiments, the branching group comprises:

wherein each j is an integer from 1 to 3; and

wherein each n is an integer from 1 to 20.

In certain embodiments, the branching group comprises:

In certain embodiments, each tether is selected from among:

wherein L is selected from a phosphorus linking group and a neutral linking group;

-   -   Z₁ is C(═O)O—R₂;     -   Z₂ is H, C₁-C₆ alkyl or substituted C₁-C₆ alky;     -   R₂ is H, C₁-C₆ alkyl or substituted C₁-C₆ alky; and     -   each m₁ is, independently, from 0 to 20 wherein at least one m₁         is greater than 0 for each tether.

In certain embodiments, each tether is selected from among:

wherein Z₂ is H or CH₃; and

each m₂ is, independently, from 0 to 20 wherein at least one m₂ is greater than 0 for each tether.

In certain embodiments, each tether is selected from among:

wherein n is from 1 to 12; and

wherein m is from 1 to 12.

In certain embodiments, at least one tether comprises ethylene glycol.

In certain embodiments, at least one tether comprises an amide. In certain embodiments, at least one tether comprises a polyamide.

In certain embodiments, at least one tether comprises an amine.

In certain embodiments, at least two tethers are different from one another. In certain embodiments, all of the tethers are the same as one another.

In certain embodiments, each tether is selected from among:

wherein each n is, independently, from 1 to 20; and

each p is from 1 to about 6.

In certain embodiments, each tether is selected from among:

In certain embodiments, each tether has the following structure:

wherein each n is, independently, from 1 to 20.

In certain embodiments, each tether has the following structure:

In certain embodiments, the tether has a structure selected from among:

wherein each n is independently, 0, 1, 2, 3, 4, 5, 6, or 7.

In certain embodiments, the tether has a structure selected from among:

In certain embodiments, the ligand is galactose.

In certain embodiments, the ligand is mannose-6-phosphate.

In certain embodiments, each ligand is selected from among:

wherein each R₁ is selected from OH and NHCOOH.

In certain embodiments, each ligand is selected from among:

In certain embodiments, each ligand has the following structure:

In certain embodiments, each ligand has the following structure:

In certain embodiments, the conjugate group comprises a cell-targeting moiety.

In certain embodiments, the conjugate group comprises a cell-targeting moiety having the following structure:

wherein each n is, independently, from 1 to 20.

In certain embodiments, the cell-targeting moiety has the following structure:

In certain embodiments, the cell-targeting moiety has the following structure:

wherein each n is, independently, from 1 to 20.

In certain embodiments, the cell-targeting moiety has the following structure:

In certain embodiments, the cell-targeting moiety comprises:

In certain embodiments, the cell-targeting moiety comprises:

In certain embodiments, the cell-targeting moiety has the following structure:

In certain embodiments, the cell-targeting moiety has the following structure:

In certain embodiments, the cell-targeting moiety comprises:

In certain embodiments, the cell-targeting moiety has the following structure

In certain embodiments, the cell-targeting moiety comprises:

In certain embodiments, the cell-targeting moiety comprises:

In certain embodiments, the cell-targeting moiety comprises:

In certain embodiments, the cell-targeting moiety has the following structure:

In certain embodiments, the cell-targeting moiety has the following structure:

In certain embodiments, the cell-targeting moiety has the following structure:

In certain embodiments, the cell-targeting moiety has the following structure:

In certain embodiments, the cell-targeting moiety has the following structure:

In certain embodiments, the cell-targeting moiety comprises:

In certain embodiments, the cell-targeting moiety comprises:

In certain embodiments, the cell-targeting moiety comprises:

In certain embodiments, the cell-targeting moiety comprises:

In certain embodiments, the cell-targeting moiety has the following structure:

In certain embodiments, the cell-targeting moiety comprises:

In certain embodiments, the cell-targeting moiety has the following structure:

In certain embodiments, the cell-targeting moiety comprises:

wherein each Y is selected from O, S, a substituted or unsubstituted C₁-C₁₀ alkyl, amino, substituted amino, azido, alkenyl or alkynyl.

In certain embodiments, the conjugate group comprises:

wherein each Y is selected from O, S, a substituted or unsubstituted C₁-C₁₀ alkyl, amino, substituted amino, azido, alkenyl or alkynyl.

In certain embodiments, the cell-targeting moiety has the following structure:

wherein each Y is selected from O, S, a substituted or unsubstituted C₁-C₁₀ alkyl, amino, substituted amino, azido, alkenyl or alkynyl.

In certain embodiments, the conjugate group comprises:

In certain embodiments, the conjugate group comprises:

In certain embodiments, the conjugate group comprises:

In certain embodiments, the conjugate group comprises:

In certain embodiments, the conjugate group comprises a cleavable moiety selected from among: a phosphodiester, an amide, or an ester.

In certain embodiments, the conjugate group comprises a phosphodiester cleavable moiety.

In certain embodiments, the conjugate group does not comprise a cleavable moiety, and wherein the conjugate group comprises a phosphorothioate linkage between the conjugate group and the oligonucleotide.

In certain embodiments, the conjugate group comprises an amide cleavable moiety.

In certain embodiments, the conjugate group comprises an ester cleavable moiety.

In certain embodiments, the compound has the following structure:

wherein each n is, independently, from 1 to 20;

Q₁₃ is H or O(CH₂)₂—OCH₃;

A is the modified oligonucleotide; and

Bx is a heterocyclic base moiety.

In certain embodiments, the compound has the following structure:

wherein each n is, independently, from 1 to 20;

Q₁₃ is H or O(CH₂)₂—OCH₃;

A is the modified oligonucleotide; and

Bx is a heterocyclic base moiety.

In certain embodiments, the compound has the following structure:

wherein each n is, independently, from 1 to 20;

Q₁₃ is H or O(CH₂)₂—OCH₃;

A is the modified oligonucleotide;

Z is H or a linked solid support; and

Bx is a heterocyclic base moiety.

In certain embodiments, the compound has the following structure:

wherein each n is, independently, from 1 to 20;

Q₁₃ is H or O(CH₂)₂—OCH₃;

A is the modified oligonucleotide;

Z is H or a linked solid support; and

Bx is a heterocyclic base moiety.

In certain embodiments, the compound has the following structure:

wherein Q₁₃ is H or O(CH₂)₂—OCH₃;

A is the modified oligonucleotide; and

Bx is a heterocyclic base moiety.

In certain embodiments, the compound has the following structure:

wherein Q₁₃ is H or O(CH₂)₂—OCH₃;

A is the modified oligonucleotide; and

Bx is a heterocyclic base moiety.

In certain embodiments, the compound has the following structure:

wherein Q₁₃ is H or O(CH₂)₂—OCH₃;

A is the modified oligonucleotide; and

Bx is a heterocyclic base moiety.

In certain embodiments, the compound has the following structure:

wherein Q₁₃ is H or O(CH₂)₂—OCH₃;

A is the modified oligonucleotide; and

Bx is a heterocyclic base moiety.

In certain embodiments, the compound has the following structure:

wherein Q₁₃ is H or O(CH₂)₂—OCH₃;

A is the modified oligonucleotide; and

Bx is a heterocyclic base moiety.

In certain embodiments, the compound has the following structure:

wherein Q₁₃ is H or O(CH₂)₂—OCH₃;

A is the modified oligonucleotide; and

Bx is a heterocyclic base moiety.

In certain embodiments, the compound has the following structure:

wherein Q₁₃ is H or O(CH₂)₂—OCH₃;

A is the modified oligonucleotide; and

Bx is a heterocyclic base moiety.

In certain embodiments, the compound has the following structure:

wherein Q₁₃ is H or O(CH₂)₂—OCH₃;

A is the modified oligonucleotide; and

Bx is a heterocyclic base moiety.

In certain embodiments, the compound has the following structure:

wherein Q₁₃ is H or O(CH₂)₂—OCH₃;

A is the modified oligonucleotide; and

Bx is a heterocyclic base moiety.

In certain embodiments, the compound has the following structure:

wherein Q₁₃ is H or O(CH₂)₂—OCH₃;

A is the modified oligonucleotide; and

Bx is a heterocyclic base moiety.

In certain embodiments, the compound has the following structure:

wherein Q₁₃ is H or O(CH₂)₂—OCH₃;

A is the modified oligonucleotide; and

Bx is a heterocyclic base moiety.

In certain embodiments, the conjugate group comprises:

wherein Q₁₃ is H or O(CH₂)₂—OCH₃;

A is the modified oligonucleotide; and

Bx is a heterocyclic base moiety.

In certain embodiments, the conjugate group comprises:

wherein Q₁₃ is H or O(CH₂)₂—OCH₃;

A is the modified oligonucleotide; and

Bx is a heterocyclic base moiety.

In certain embodiments, the conjugate group comprises:

wherein Q₁₃ is H or O(CH₂)₂—OCH₃;

A is the modified oligonucleotide; and

Bx is a heterocyclic base moiety.

In certain embodiments, B_(x) is selected from among from adenine, guanine, thymine, uracil, or cytosine, or 5-methyl cytosine. In certain embodiments, B_(x) is adenine. In certain embodiments, B_(x) is thymine. In certain embodiments, Q₁₃ is O(CH₂)₂—OCH₃. In certain embodiments, Q₁₃ is H.

Certain embodiments of the invention provide a prodrug comprising the compositions or compounds disclosed herein. Certain embodiments provide methods of using the conjugated antisense compounds and compositions described herein for inhibiting ANGPTL3 expression. In certain embodiments, the conjugated antisense compounds or compositions inhibit ANGPTL3 by at least 5%, at least 10%, at least 20%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95%. In a preferred embodiment, the antisense compound comprising a modified oligonucleotide and a conjugate group decreases ANGPTL3 by at least 50%. In a preferred embodiment, the antisense compound comprising a modified oligonucleotide and a conjugate group decreases ANGPTL3 by at least 55%. In a preferred embodiment the antisense compound comprising a modified oligonucleotide and a conjugate group decreases ANGPTL3 by at least 60%. In a preferred embodiment, the antisense compound comprising a modified oligonucleotide and a conjugate group decreases ANGPTL3 by at least 65%. In a preferred embodiment, the antisense compound comprising a modified oligonucleotide and a conjugate group decreases ANGPTL3 by at least 70%. In a preferred embodiment, the antisense compound comprising a modified oligonucleotide and a conjugate group decreases ANGPTL3 by at least 75%. In a preferred embodiment, the antisense compound comprising a modified oligonucleotide and a conjugate group decreases ANGPTL3 by at least 80%. In a preferred embodiment, the antisense compound comprising a modified oligonucleotide and a conjugate group decreases ANGPTL3 by at least 85%. In a preferred embodiment, the antisense compound comprising a modified oligonucleotide and a conjugate group decreases ANGPTL3 by at least 90%. In a preferred embodiment, the antisense compound comprising a modified oligonucleotide and a conjugate group decreases ANGPTL3 by at least 95%.

In certain embodiments, the conjugated antisense compounds or compositions disclosed herein have an IC₅₀ of less than 20 μM, less than 10 μM, less than 8 μM, less than 5 μM, less than 2 μM, less than 1 μM, or less than 0.8 μM, when tested human cells, for example, in the Hep3B cell line as described in Examples 2-3 and 7-10.

In certain embodiments, the conjugated antisense compounds or compositions disclosed herein are efficacious by virtue of having a viscosity of less than 40 cP, less than 35 cP, less than 30 cP, less than 25 cP, less than 20 cP or less than 15 cP when measured by the parameters as described in Example 13.

In certain embodiments, the conjugated antisense compounds or compositions disclosed herein are highly tolerable, as demonstrated by the in vivo tolerability measurements described in the examples. In certain embodiments, the conjugated antisense compounds as described herein are highly tolerable, as demonstrated by having an increase in ALT and/or AST value of no more than 4 fold, 3 fold, 2 fold or 1.5 fold over saline treated animals.

Certain embodiments disclosed herein provide a salt of the conjugated antisense compounds disclosed herein. In certain embodiments, the compounds or compositions disclosed herein comprise a salt of the modified oligonucleotide with the conjugate group.

In certain embodiments, the conjugated antisense compounds or compositions disclosed herein further comprise a pharmaceutically acceptable carrier or diluent.

In certain embodiments, the animal is a human.

Certain embodiments disclosed herein provide methods comprising administering to an animal the conjugated antisense compounds or compositions disclosed herein. In certain embodiments, administering the conjugated antisense compound or composition is therapeutic. In certain embodiments, administering the conjugated antisense compound or composition treats, prevents, or slows progression of a disease related to ANGPTL3. In certain embodiments, the disease is related to elevated ANGPTL3. In certain embodiments, administering the conjugated antisense compound or composition prevents, treats, ameliorates, or slows progression of a cardiovascular and/or metabolic disease.

Certain embodiments disclosed herein provide methods for treating a human with a cardiovascular and/or metabolic disease comprising identifying a human with cardiovascular and/or metabolic disease and administering to the human a therapeutically effective amount of any of the conjugated antisense compounds or compositions disclosed herein, so as to treat the human for cardiovascular and/or metabolic disease.

Certain embodiments provide conjugated antisense compounds and compositions described herein for use in therapy. In certain embodiments, the therapy is used in treating, preventing, or slowing progression of a disease related to ANGPTL3. In certain embodiments, the therapy is used in treating, preventing, or slowing progression of a disease related to elevated ANGPTL3.

In certain embodiments, the disease is a cardiovascular and/or metabolic disease, disorder or condition. In certain embodiments, the metabolic and/or cardiovascular disease includes, but is not limited to, obesity, diabetes, insulin resistance, atherosclerosis, dyslipidemia, lipodystrophy, coronary heart disease, non-alcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH) hyperfattyacidemia or metabolic syndrome, or a combination thereof. The dyslipidemia can be hyperlipidemia. The hyperlipidemia can be combined hyperlipidemia (CHL), hypercholesterolemia, hypertriglyceridemia, or both hypercholesterolemia and hypertriglyceridemia. The combined hyperlipidemia can be familial or non-familial. The hypercholesterolemia can be familial homozygous hypercholesterolemia (HoFH), familial heterozygous hypercholesterolemia (HeFH). The hypertriglyceridemia can be familial chylomicronemia syndrome (FCS) or hyperlipoproteinemia Type IV. The NAFLD can be hepatic steatosis or steatohepatitis. The diabetes can be type 2 diabetes or type 2 diabetes with dyslipidemia. The insulin resistance can be insulin resistance with dyslipidemia.

In certain embodiments, the conjugated antisense compounds or compositions disclosed herein are designated as a first agent and the methods or uses disclosed herein further comprise administering a second agent. In certain embodiments, the first agent and the second agent are co-administered. In certain embodiments the first agent and the second agent are co-administered sequentially or concomitantly.

In certain embodiments, the second agent is a glucose-lowering agent. The glucose lowering agent can include, but is not limited to, a therapeutic lifestyle change, PPAR agonist, a dipeptidyl peptidase (IV) inhibitor, a GLP-1 analog, insulin or an insulin analog, an insulin secretagogue, a SGLT2 inhibitor, a human amylin analog, a biguanide, an alpha-glucosidase inhibitor, or a combination thereof. The glucose-lowering agent can include, but is not limited to metformin, sulfonylurea, rosiglitazone, meglitinide, thiazolidinedione, alpha-glucosidase inhibitor or a combination thereof. The sulfonylurea can be acetohexamide, chlorpropamide, tolbutamide, tolazamide, glimepiride, a glipizide, a glyburide, or a gliclazide. The meglitinide can be nateglinide or repaglinide. The thiazolidinedione can be pioglitazone or rosiglitazone. The alpha-glucosidase can be acarbose or miglitol.

In certain embodiments, the second agent is a lipid-lowering therapy. In certain embodiments the lipid lowering therapy can include, but is not limited to, a therapeutic lifestyle change, HMG-CoA reductase inhibitor, cholesterol absorption inhibitor, MTP inhibitor (e.g., a small molecule, polypeptide, antibody or antisense compound targeted to MTP), ApoB inhibitor (e.g., a small molecule, polypeptide, antibody or antisense compound targeted to ApoB), ApoC3 inhibitor (e.g., a small molecule, polypeptide, antibody or antisense compound targeted to ApoC3), PCSK9 inhibitor (e.g., a small molecule, polypeptide, antibody or antisense compound targeted to PCSK9), CETP inhibitor (e.g., a small molecule, polypeptide, antibody or antisense compound targeted to CETP), fibrate, beneficial oil (e.g., krill or fish oils (e.g., Vascepa®), flaxseed oil, or other oils rich in omega-3 fatty acids such as α-linolenic acid (ALA), docosahexaenoic acid (DHA) or eicosapentaenoic acid (EPA)), or any combination thereof. The HMG-CoA reductase inhibitor can be atorvastatin, rosuvastatin, fluvastatin, lovastatin, pravastatin, or simvastatin. The cholesterol absorption inhibitor can be ezetimibe. The fibrate can be fenofibrate, bezafibrate, ciprofibrate, clofibrate, gemfibrozil and the like.

In certain embodiments, administration comprises parenteral administration. In certain embodiments, administration comprises subcutaneous administration.

In certain embodiments, administering a conjugated antisense compound disclosed herein results in a reduction of lipid levels, including triglyceride levels, cholesterol levels, insulin resistance, glucose levels or a combination thereof. One or more of the levels can be independently reduced by at least 5%, at least 10%, at least 20%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95%. Administering the conjugated antisense compound can result in improved insulin sensitivity or hepatic insulin sensitivity. Administering the conjugated antisense compound disclosed herein can result in a reduction in atherosclerotic plaques, obesity, glucose, lipids, glucose resistance, cholesterol, or improvement in insulin sensitivity or any combination thereof.

Certain embodiments provide the use of a conjugated antisense compound as described herein in the manufacture of a medicament for treating, ameliorating, delaying or preventing one or more of a disease related to ANGPTL3. Certain embodiments provide the use of a conjugated antisense compound as described herein in the manufacture of a medicament for treating, ameliorating, delaying or preventing one or more of a metabolic disease or a cardiovascular disease.

Certain embodiments provide a kit for treating, preventing, or ameliorating one or more of a metabolic disease or a cardiovascular disease as described herein wherein the kit comprises: a) a conjugated antisense compound as described herein; and optionally b) an additional agent or therapy as described herein. The kit can further include instructions or a label for using the kit to treat, prevent, or ameliorate one or more of a metabolic disease or a cardiovascular disease.

Antisense Compounds

Oligomeric compounds include, but are not limited to, oligonucleotides, oligonucleosides, oligonucleotide analogs, oligonucleotide mimetics, antisense compounds, antisense oligonucleotides, and siRNAs. An oligomeric compound can be “antisense” to a target nucleic acid, meaning that is capable of undergoing hybridization to a target nucleic acid through hydrogen bonding.

In certain embodiments, an antisense compound has a nucleobase sequence that, when written in the 5′ to 3′ direction, comprises the reverse complement of the target segment of a target nucleic acid to which it is targeted. In certain such embodiments, an antisense oligonucleotide has a nucleobase sequence that, when written in the 5′ to 3′ direction, comprises the reverse complement of the target segment of a target nucleic acid to which it is targeted.

In certain embodiments, an antisense compound targeted to ANGPTL3 nucleic acid is 10 to 30 nucleotides in length. In other words, antisense compounds are from 10 to 30 linked nucleobases. In other embodiments, the antisense compound comprises a modified oligonucleotide consisting of 8 to 80, 10 to 80, 12 to 50, 12 to 30, 15 to 30, 18 to 24, 19 to 22, or 20 linked nucleobases. In certain such embodiments, the antisense compound comprises a modified oligonucleotide consisting of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 linked nucleobases in length, or a range defined by any two of the above values.

In certain embodiments, the antisense compound comprises a shortened or truncated modified oligonucleotide. The shortened or truncated modified oligonucleotide can have a single nucleoside deleted from the 5′ end (5′ truncation), or alternatively from the 3′ end (3′ truncation). A shortened or truncated oligonucleotide can have two or more nucleosides deleted from the 5′ end, or alternatively can have two or more nucleosides deleted from the 3′ end. Alternatively, the deleted nucleosides can be dispersed throughout the modified oligonucleotide, for example, in an antisense compound having one or more nucleoside deleted from the 5′ end and one or more nucleoside deleted from the 3′ end.

When a single additional nucleoside is present in a lengthened oligonucleotide, the additional nucleoside can be located at the 5′, 3′ end or central portion of the oligonucleotide. When two or more additional nucleosides are present, the added nucleosides can be adjacent to each other, for example, in an oligonucleotide having two nucleosides added to the 5′ end (5′ addition), or alternatively to the 3′ end (3′ addition) or the central portion, of the oligonucleotide. Alternatively, the added nucleoside can be dispersed throughout the antisense compound, for example, in an oligonucleotide having one or more nucleoside added to the 5′ end, one or more nucleoside added to the 3′ end, and/or one or more nucleoside added to the central portion.

It is possible to increase or decrease the length of an antisense compound, such as an antisense oligonucleotide, and/or introduce mismatch bases without eliminating activity. For example, in Woolf et al. (Proc. Natl. Acad. Sci. USA 89:7305-7309, 1992), a series of antisense oligonucleotides 13-25 nucleobases in length were tested for their ability to induce cleavage of a target RNA in an oocyte injection model. Antisense oligonucleotides 25 nucleobases in length with 8 or 11 mismatch bases near the ends of the antisense oligonucleotides were able to direct specific cleavage of the target mRNA, albeit to a lesser extent than the antisense oligonucleotides that contained no mismatches. Similarly, target specific cleavage was achieved using 13 nucleobase antisense oligonucleotides, including those with 1 or 3 mismatches.

Gautschi et al (J. Natl. Cancer Inst. 93:463-471, March 2001) demonstrated the ability of an oligonucleotide having 100% complementarity to the bcl-2 mRNA and having 3 mismatches to the bcl-xL mRNA to reduce the expression of both bcl-2 and bcl-xL in vitro and in vivo. Furthermore, this oligonucleotide demonstrated potent anti-tumor activity in vivo.

Maher and Dolnick (Nuc. Acid. Res. 16:3341-3358, 1988) tested a series of tandem 14 nucleobase antisense oligonucleotides, and a 28 and 42 nucleobase antisense oligonucleotides comprised of the sequence of two or three of the tandem antisense oligonucleotides, respectively, for their ability to arrest translation of human DHFR in a rabbit reticulocyte assay. Each of the three 14 nucleobase antisense oligonucleotides alone was able to inhibit translation, albeit at a more modest level than the 28 or 42 nucleobase antisense oligonucleotides.

Certain Antisense Compound Motifs and Mechanisms

In certain embodiments, antisense compounds have chemically modified subunits arranged in patterns, or motifs, to confer to the antisense compounds properties such as enhanced inhibitory activity, increased binding affinity for a target nucleic acid, or resistance to degradation by in vivo nucleases. Chimeric antisense compounds typically contain at least one region modified so as to confer increased resistance to nuclease degradation, increased cellular uptake, increased binding affinity for the target nucleic acid, and/or increased inhibitory activity. A second region of a chimeric antisense compound may confer another desired property e.g., serve as a substrate for the cellular endonuclease RNase H, which cleaves the RNA strand of an RNA:DNA duplex.

Antisense activity may result from any mechanism involving the hybridization of the antisense compound (e.g., oligonucleotide) with a target nucleic acid, wherein the hybridization ultimately results in a biological effect. In certain embodiments, the amount and/or activity of the target nucleic acid is modulated. In certain embodiments, the amount and/or activity of the target nucleic acid is reduced. In certain embodiments, hybridization of the antisense compound to the target nucleic acid ultimately results in target nucleic acid degradation. In certain embodiments, hybridization of the antisense compound to the target nucleic acid does not result in target nucleic acid degradation. In certain such embodiments, the presence of the antisense compound hybridized with the target nucleic acid (occupancy) results in a modulation of antisense activity. In certain embodiments, antisense compounds having a particular chemical motif or pattern of chemical modifications are particularly suited to exploit one or more mechanisms. In certain embodiments, antisense compounds function through more than one mechanism and/or through mechanisms that have not been elucidated. Accordingly, the antisense compounds described herein are not limited by particular mechanism.

Antisense mechanisms include, without limitation, RNase H mediated antisense; RNAi mechanisms, which utilize the RISC pathway and include, without limitation, siRNA, ssRNA and microRNA mechanisms; and occupancy based mechanisms. Certain antisense compounds may act through more than one such mechanism and/or through additional mechanisms.

RNase H-Mediated Antisense

In certain embodiments, antisense activity results at least in part from degradation of target RNA by RNase H. RNase H is a cellular endonuclease that cleaves the RNA strand of an RNA:DNA duplex. It is known in the art that single-stranded antisense compounds which are “DNA-like” elicit RNase H activity in mammalian cells. Accordingly, antisense compounds comprising at least a portion of DNA or DNA-like nucleosides may activate RNase H, resulting in cleavage of the target nucleic acid. In certain embodiments, antisense compounds that utilize RNase H comprise one or more modified nucleosides. In certain embodiments, such antisense compounds comprise at least one block of 1-8 modified nucleosides. In certain such embodiments, the modified nucleosides do not support RNase H activity. In certain embodiments, such antisense compounds are gapmers, as described herein. In certain such embodiments, the gap of the gapmer comprises DNA nucleosides. In certain such embodiments, the gap of the gapmer comprises DNA-like nucleosides. In certain such embodiments, the gap of the gapmer comprises DNA nucleosides and DNA-like nucleosides.

Certain antisense compounds having a gapmer motif are considered chimeric antisense compounds. In a gapmer an internal region having a plurality of nucleotides that supports RNaseH cleavage is positioned between external regions having a plurality of nucleotides that are chemically distinct from the nucleosides of the internal region. In the case of an antisense oligonucleotide having a gapmer motif, the gap segment generally serves as the substrate for endonuclease cleavage, while the wing segments comprise modified nucleosides. In certain embodiments, the regions of a gapmer are differentiated by the types of sugar moieties comprising each distinct region. The types of sugar moieties that are used to differentiate the regions of a gapmer may in some embodiments include β-D-ribonucleosides, β-D-deoxyribonucleosides, 2′-modified nucleosides (such 2′-modified nucleosides may include 2′-MOE and 2′-O—CH₃, among others), and bicyclic sugar modified nucleosides (such bicyclic sugar modified nucleosides may include those having a constrained ethyl). In certain embodiments, nucleosides in the wings may include several modified sugar moieties, including, for example 2′-MOE and bicyclic sugar moieties such as constrained ethyl or LNA. In certain embodiments, wings may include several modified and unmodified sugar moieties. In certain embodiments, wings may include various combinations of 2′-MOE nucleosides, bicyclic sugar moieties such as constrained ethyl nucleosides or LNA nucleosides, and 2′-deoxynucleosides.

Each distinct region may comprise uniform sugar moieties, variant, or alternating sugar moieties. The wing-gap-wing motif is frequently described as “X—Y—Z”, where “X” represents the length of the 5′-wing, “Y” represents the length of the gap, and “Z” represents the length of the 3′-wing. “X” and “Z” may comprise uniform, variant, or alternating sugar moieties. In certain embodiments, “X” and “Y” may include one or more 2′-deoxynucleosides. “Y” may comprise 2′-deoxynucleosides. As used herein, a gapmer described as “X—Y—Z” has a configuration such that the gap is positioned immediately adjacent to each of the 5′-wing and the 3′ wing. Thus, no intervening nucleotides exist between the 5′-wing and gap, or the gap and the 3′-wing. Any of the antisense compounds described herein can have a gapmer motif. In certain embodiments, “X” and “Z” are the same; in other embodiments they are different. In certain embodiments, “Y” is between 8 and 15 nucleosides. X, Y, or Z can be any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30 or more nucleosides.

In certain embodiments, the antisense compound targeted to an ANGPTL3 nucleic acid has a gapmer motif in which the gap consists of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 linked nucleosides.

In certain embodiments, the antisense oligonucleotide has a sugar motif described by Formula A as follows: (J)_(m)-(B)_(n)-(J)_(p)-(B)_(r)-(A)_(t)-(D)_(g)-(A)_(v)-(B)_(w)-(J)_(x)-(B)_(y)-(J)_(z)

wherein:

each A is independently a 2′-substituted nucleoside;

each B is independently a bicyclic nucleoside;

each J is independently either a 2′-substituted nucleoside or a 2′-deoxynucleoside;

each D is a 2′-deoxynucleoside;

m is 0-4; n is 0-2; p is 0-2; r is 0-2; t is 0-2; v is 0-2; w is 0-4; x is 0-2; y is 0-2; z is 0-4; g is 6-14; provided that:

-   -   at least one of m, n, and r is other than 0;     -   at least one of w and y is other than 0;     -   the sum of m, n, p, r, and t is from 2 to 5; and the sum of v,         w, x, y, and z is from 2 to 5.

RNAi Compounds

In certain embodiments, antisense compounds are interfering RNA compounds (RNAi), which include double-stranded RNA compounds (also referred to as short-interfering RNA or siRNA) and single-stranded RNAi compounds (or ssRNA). Such compounds work at least in part through the RISC pathway to degrade and/or sequester a target nucleic acid (thus, include microRNA/microRNA-mimic compounds). In certain embodiments, antisense compounds comprise modifications that make them particularly suited for such mechanisms.

1. ssRNA Compounds

In certain embodiments, antisense compounds including those particularly suited for use as single-stranded RNAi compounds (ssRNA) comprise a modified 5′-terminal end. In certain such embodiments, the 5′-terminal end comprises a modified phosphate moiety. In certain embodiments, such modified phosphate is stabilized (e.g., resistant to degradation/cleavage compared to unmodified 5′-phosphate). In certain embodiments, such 5′-terminal nucleosides stabilize the 5′-phosphorous moiety. Certain modified 5′-terminal nucleosides may be found in the art, for example in WO/2011/139702.

In certain embodiments, the 5′-nucleoside of an ssRNA compound has Formula IIc:

wherein:

T₁ is an optionally protected phosphorus moiety;

T₂ is an internucleoside linking group linking the compound of Formula IIc to the oligomeric compound;

A has one of the formulas:

Q₁ and Q₂ are each, independently, H, halogen, C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₁-C₆ alkoxy, substituted C₁-C₆ alkoxy, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl, substituted C₂-C₆ alkynyl or N(R₃)(R₄);

Q₃ is O, S, N(R₅) or C(R₆)(R₇);

each R₃, R₄, R₅, R₆ and R₇ is, independently, H, C₁-C₆ alkyl, substituted C₁-C₆ alkyl or C₁-C₆ alkoxy;

M₃ is O, S, NR₁₄, C(R₁₅)(R₁₆), C(R₁₅)(R₁₆)C(R₁₇)(R₁₈), C(R₁₅)═C(R₁₇), OC(R₁₅)(R₁₆) or OC(R₁₅)(Bx₂);

R₁₄ is H, C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₁-C₆ alkoxy, substituted C₁-C₆ alkoxy, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl or substituted C₂-C₆ alkynyl;

R₁₅, R₁₆, R₁₇ and R₁₈ are each, independently, H, halogen, C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₁-C₆ alkoxy, substituted C₁-C₆ alkoxy, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl or substituted C₂-C₆ alkynyl;

Bx₁ is a heterocyclic base moiety;

or if Bx₂ is present then Bx₂ is a heterocyclic base moiety and Bx₁ is H, halogen, C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₁-C₆ alkoxy, substituted C₁-C₆ alkoxy, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl or substituted C₂-C₆ alkynyl;

J₄, J₅, J₆ and J₇ are each, independently, H, halogen, C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₁-C₆ alkoxy, substituted C₁-C₆ alkoxy, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl or substituted C₂-C₆ alkynyl;

or J₄ forms a bridge with one of J₅ or J₇ wherein said bridge comprises from 1 to 3 linked biradical groups selected from O, S, NR₁₉, C(R₂₀)(R₂₁), C(R₂₀)═C(R₂₁), C[═C(R₂₀)(R₂₁)] and C(═O) and the other two of J₅, J₆ and J₇ are each, independently, H, halogen, C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₁-C₆ alkoxy, substituted C₁-C₆ alkoxy, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl or substituted C₂-C₆ alkynyl;

each R₁₉, R₂₀ and R₂₁ is, independently, H, C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₁-C₆ alkoxy, substituted C₁-C₆ alkoxy, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl or substituted C₂-C₆ alkynyl;

G is H, OH, halogen or O—[C(R₈)(R₉)]_(n)—[(C═O)_(m)—X₁]_(j)—Z;

each R₈ and R₉ is, independently, H, halogen, C₁-C₆ alkyl or substituted C₁-C₆ alkyl;

X₁ is O, S or N(E₁);

Z is H, halogen, C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl, substituted C₂-C₆ alkynyl or N(E₂)(E₃);

E₁, E₂ and E₃ are each, independently, H, C₁-C₆ alkyl or substituted C₁-C₆ alkyl;

n is from 1 to about 6;

m is 0 or 1;

j is 0 or 1;

each substituted group comprises one or more optionally protected substituent groups independently selected from halogen, OJ₁, N(J₁)(J₂), =NJ₁, SJ₁, N₃, CN, OC(═X₂)J₁, OC(═X₂)N(J₁)(J₂) and C(═X₂)N(J₁)(J₂);

X₂ is O, S or NJ₃;

each J₁, J₂ and J₃ is, independently, H or C₁-C₆ alkyl;

when j is 1 then Z is other than halogen or N(E₂)(E₃); and

wherein said oligomeric compound comprises from 8 to 40 monomeric subunits and is hybridizable to at least a portion of a target nucleic acid.

In certain embodiments, M₃ is O, CH═CH, OCH₂ or OC(H)(Bx₂). In certain embodiments, M₃ is O.

In certain embodiments, J₄, J₅, J₆ and J₇ are each H. In certain embodiments, J₄ forms a bridge with one of J₅ or J₇.

In certain embodiments, A has one of the formulas:

wherein:

Q₁ and Q₂ are each, independently, H, halogen, C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₁-C₆ alkoxy or substituted C₁-C₆ alkoxy. In certain embodiments, Q₁ and Q₂ are each H. In certain embodiments, Q₁ and Q₂ are each, independently, H or halogen. In certain embodiments, Q₁ and Q₂ is H and the other of Q₁ and Q₂ is F, CH₃ or OCH₃.

In certain embodiments, T₁ has the formula:

wherein:

R_(a) and R_(c) are each, independently, protected hydroxyl, protected thiol, C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₁-C₆ alkoxy, substituted C₁-C₆ alkoxy, protected amino or substituted amino; and

R_(b) is O or S. In certain embodiments, R_(b) is O and R_(a) and R_(c) are each, independently, OCH₃, OCH₂CH₃ or CH(CH₃)₂.

In certain embodiments, G is halogen, OCH₃, OCH₂F, OCHF₂, OCF₃, OCH₂CH₃, O(CH₂)₂F, OCH₂CHF₂, OCH₂CF₃, OCH₂—CH═CH₂, O(CH₂)₂—OCH₃, O(CH₂)₂—SCH₃, O(CH₂)₂—OCF₃, O(CH₂)₃—N(R₁₀)(R₁₁), O(CH₂)₂—ON(R₁₀)(R₁₁), O(CH₂)₂—O(CH₂)₂—N(R₁₀)(R₁), OCH₂C(═O)—N(R₁₀)(R₁), OCH₂C(═O)—N(R₁₂)—(CH₂)₂—N(R₁₀)(R₁) or O(CH₂)₂—N(R₁₂)—C(═NR₁₃)[N(R₁₀)(R₁₁)] wherein R₁₀, R₁₁, R₁₂ and R₁₃ are each, independently, H or C₁-C₆ alkyl. In certain embodiments, G is halogen, OCH₃, OCF₃, OCH₂CH₃, OCH₂CF₃, OCH₂—CH═CH₂, O(CH₂)₂—OCH₃, O(CH₂)₂—O(CH₂)₂—N(CH₃)₂, OCH₂C(═O)—N(H)CH₃, OCH₂C(═O)—N(H)—(CH₂)₂—N(CH₃)₂ or OCH₂—N(H)—C(═NH)NH₂. In certain embodiments, G is F, OCH₃ or O(CH₂)₂—OCH₃. In certain embodiments, G is O(CH₂)₂—OCH₃.

In certain embodiments, the 5′-terminal nucleoside has Formula IIe:

In certain embodiments, antisense compounds, including those particularly suitable for ssRNA comprise one or more type of modified sugar moieties and/or naturally occurring sugar moieties arranged along an oligonucleotide or region thereof in a defined pattern or sugar modification motif. Such motifs may include any of the sugar modifications discussed herein and/or other known sugar modifications.

In certain embodiments, the oligonucleotides comprise or consist of a region having uniform sugar modifications. In certain such embodiments, each nucleoside of the region comprises the same RNA-like sugar modification. In certain embodiments, each nucleoside of the region is a 2′-F nucleoside. In certain embodiments, each nucleoside of the region is a 2′-OMe nucleoside. In certain embodiments, each nucleoside of the region is a 2′-MOE nucleoside. In certain embodiments, each nucleoside of the region is a cEt nucleoside. In certain embodiments, each nucleoside of the region is an LNA nucleoside. In certain embodiments, the uniform region constitutes all or essentially all of the oligonucleotide. In certain embodiments, the region constitutes the entire oligonucleotide except for 1-4 terminal nucleosides.

In certain embodiments, oligonucleotides comprise one or more regions of alternating sugar modifications, wherein the nucleosides alternate between nucleotides having a sugar modification of a first type and nucleotides having a sugar modification of a second type. In certain embodiments, nucleosides of both types are RNA-like nucleosides. In certain embodiments the alternating nucleosides are selected from: 2′-OMe, 2′-F, 2′-MOE, LNA, and cEt. In certain embodiments, the alternating modificatios are 2′-F and 2′-OMe. Such regions may be contiguous or may be interupted by differently modified nucleosides or conjugated nucleosides.

In certain embodiments, the alternating region of alternating modifications each consist of a single nucleoside (i.e., the patern is (AB)_(x)A_(y) wheren A is a nucleoside having a sugar modification of a first type and B is a nucleoside having a sugar modification of a second type; x is 1-20 and y is 0 or 1). In certan embodiments, one or more alternating regions in an alternating motif includes more than a single nucleoside of a type. For example, oligonucleotides may include one or more regions of any of the following nucleoside motifs:

AABBAA;

ABBABB;

AABAAB;

ABBABAABB;

ABABAA;

AABABAB;

ABABAA;

ABBAABBABABAA;

BABBAABBABABAA; or

ABABBAABBABABAA;

wherein A is a nucleoside of a first type and B is a nucleoside of a second type. In certain embodiments, A and B are each selected from 2′-F, 2′-OMe, BNA, and MOE.

In certain embodiments, oligonucleotides having such an alternating motif also comprise a modified 5′ terminal nucleoside, such as those of formula IIc or lie.

In certain embodiments, oligonucleotides comprise a region having a 2-2-3 motif. Such regions comprises the following motif: -(A)₂-(B)_(x)-(A)₂-(C)_(y)-(A)₃-

wherein: A is a first type of modified nucleoside;

B and C, are nucleosides that are differently modified than A, however, B and C may have the same or different modifications as one another;

x and y are from 1 to 15.

In certain embodiments, A is a 2′-OMe modified nucleoside. In certain embodiments, B and C are both 2′-F modified nucleosides. In certain embodiments, A is a 2′-OMe modified nucleoside and B and C are both 2′-F modified nucleosides.

In certain embodiments, oligonucleosides have the following sugar motif: 5′-(Q)-(AB)_(x)A_(y)-(D)_(z) wherein:

Q is a nucleoside comprising a stabilized phosphate moiety. In certain embodiments, Q is a nucleoside having Formula IIc or IIe;

A is a first type of modified nucleoside;

B is a second type of modified nucleoside;

D is a modified nucleoside comprising a modification different from the nucleoside adjacent to it. Thus, if y is 0, then D must be differently modified than B and if y is 1, then D must be differently modified than A. In certain embodiments, D differs from both A and B.

X is 5-15;

Y is 0 or 1;

Z is 0-4.

In certain embodiments, oligonucleosides have the following sugar motif: 5′-(Q)-(A)_(x)-(D)_(z) wherein:

Q is a nucleoside comprising a stabilized phosphate moiety. In certain embodiments, Q is a nucleoside having Formula IIc or IIe;

A is a first type of modified nucleoside;

D is a modified nucleoside comprising a modification different from A.

X is 11-30;

Z is 0-4.

In certain embodiments A, B, C, and D in the above motifs are selected from: 2′-OMe, 2′-F, 2′-MOE, LNA, and cEt. In certain embodiments, D represents terminal nucleosides. In certain embodiments, such terminal nucleosides are not designed to hybridize to the target nucleic acid (though one or more might hybridize by chance). In certiain embodiments, the nucleobase of each D nucleoside is adenine, regardless of the identity of the nucleobase at the corresponding position of the target nucleic acid. In certain embodiments the nucleobase of each D nucleoside is thymine.

In certain embodiments, antisense compounds, including those particularly suited for use as ssRNA comprise modified internucleoside linkages arranged along the oligonucleotide or region thereof in a defined pattern or modified internucleoside linkage motif. In certain embodiments, oligonucleotides comprise a region having an alternating internucleoside linkage motif. In certain embodiments, oligonucleotides comprise a region of uniformly modified internucleoside linkages. In certain such embodiments, the oligonucleotide comprises a region that is uniformly linked by phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide is uniformly linked by phosphorothioate internucleoside linkages. In certain embodiments, each internucleoside linkage of the oligonucleotide is selected from phosphodiester and phosphorothioate. In certain embodiments, each internucleoside linkage of the oligonucleotide is selected from phosphodiester and phosphorothioate and at least one internucleoside linkage is phosphorothioate.

In certain embodiments, the oligonucleotide comprises at least 6 phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least 8 phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least 10 phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least one block of at least 6 consecutive phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least one block of at least 8 consecutive phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least one block of at least 10 consecutive phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least one block of at least one 12 consecutive phosphorothioate internucleoside linkages. In certain such embodiments, at least one such block is located at the 3′ end of the oligonucleotide. In certain such embodiments, at least one such block is located within 3 nucleosides of the 3′ end of the oligonucleotide.

Oligonucleotides having any of the various sugar motifs described herein, may have any linkage motif. For example, the oligonucleotides, including but not limited to those described above, may have a linkage motif selected from non-limiting the table below:

5′ most linkage Central region 3′-region PS Alternating PO/PS 6 PS PS Alternating PO/PS 7 PS PS Alternating PO/PS 8 PS

2. siRNA Compounds

In certain embodiments, antisense compounds are double-stranded RNAi compounds (siRNA). In such embodiments, one or both strands may comprise any modification motif described above for ssRNA. In certain embodiments, ssRNA compounds may be unmodified RNA. In certain embodiments, siRNA compounds may comprise unmodified RNA nucleosides, but modified internucleoside linkages.

Several embodiments relate to double-stranded compositions wherein each strand comprises a motif defined by the location of one or more modified or unmodified nucleosides. In certain embodiments, compositions are provided comprising a first and a second oligomeric compound that are fully or at least partially hybridized to form a duplex region and further comprising a region that is complementary to and hybridizes to a nucleic acid target. It is suitable that such a composition comprise a first oligomeric compound that is an antisense strand having full or partial complementarity to a nucleic acid target and a second oligomeric compound that is a sense strand having one or more regions of complementarity to and forming at least one duplex region with the first oligomeric compound.

The compositions of several embodiments modulate gene expression by hybridizing to a nucleic acid target resulting in loss of its normal function. In some embodiments, the target nucleic acid is ANGPTL3. In certain embodiment, the degradation of the targeted ANGPTL3 is facilitated by an activated RISC complex that is formed with compositions disclosed herein.

Several embodiments are directed to double-stranded compositions wherein one of the strands is useful in, for example, influencing the preferential loading of the opposite strand into the RISC (or cleavage) complex. The compositions are useful for targeting selected nucleic acid molecules and modulating the expression of one or more genes. In some embodiments, the compositions of the present invention hybridize to a portion of a target RNA resulting in loss of normal function of the target RNA.

Certain embodiments are drawn to double-stranded compositions wherein both the strands comprises a hemimer motif, a fully modified motif, a positionally modified motif or an alternating motif. Each strand of the compositions of the present invention can be modified to fulfil a particular role in for example the siRNA pathway. Using a different motif in each strand or the same motif with different chemical modifications in each strand permits targeting the antisense strand for the RISC complex while inhibiting the incorporation of the sense strand. Within this model, each strand can be independently modified such that it is enhanced for its particular role. The antisense strand can be modified at the 5′-end to enhance its role in one region of the RISC while the 3′-end can be modified differentially to enhance its role in a different region of the RISC.

The double-stranded oligonucleotide molecules can be a double-stranded polynucleotide molecule comprising self-complementary sense and antisense regions, wherein the antisense region comprises nucleotide sequence that is complementary to nucleotide sequence in a target nucleic acid molecule or a portion thereof and the sense region having nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof. The double-stranded oligonucleotide molecules can be assembled from two separate oligonucleotides, where one strand is the sense strand and the other is the antisense strand, wherein the antisense and sense strands are self-complementary (i.e. each strand comprises nucleotide sequence that is complementary to nucleotide sequence in the other strand; such as where the antisense strand and sense strand form a duplex or double-stranded structure, for example wherein the double-stranded region is about 15 to about 30, e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 base pairs; the antisense strand comprises nucleotide sequence that is complementary to nucleotide sequence in a target nucleic acid molecule or a portion thereof and the sense strand comprises nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof (e.g., about 15 to about 25 or more nucleotides of the double-stranded oligonucleotide molecule are complementary to the target nucleic acid or a portion thereof). Alternatively, the double-stranded oligonucleotide is assembled from a single oligonucleotide, where the self-complementary sense and antisense regions of the siRNA are linked by means of a nucleic acid based or non-nucleic acid-based linker(s).

The double-stranded oligonucleotide can be a polynucleotide with a duplex, asymmetric duplex, hairpin or asymmetric hairpin secondary structure, having self-complementary sense and antisense regions, wherein the antisense region comprises nucleotide sequence that is complementary to nucleotide sequence in a separate target nucleic acid molecule or a portion thereof and the sense region having nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof. The double-stranded oligonucleotide can be a circular single-stranded polynucleotide having two or more loop structures and a stem comprising self-complementary sense and antisense regions, wherein the antisense region comprises nucleotide sequence that is complementary to nucleotide sequence in a target nucleic acid molecule or a portion thereof and the sense region having nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof, and wherein the circular polynucleotide can be processed either in vivo or in vitro to generate an active siRNA molecule capable of mediating RNAi.

In certain embodiments, the double-stranded oligonucleotide comprises separate sense and antisense sequences or regions, wherein the sense and antisense regions are covalently linked by nucleotide or non-nucleotide linkers molecules as is known in the art, or are alternately non-covalently linked by ionic interactions, hydrogen bonding, van der waals interactions, hydrophobic interactions, and/or stacking interactions. In certain embodiments, the double-stranded oligonucleotide comprises nucleotide sequence that is complementary to nucleotide sequence of a target gene. In another embodiment, the double-stranded oligonucleotide interacts with nucleotide sequence of a target gene in a manner that causes inhibition of expression of the target gene.

As used herein, double-stranded oligonucleotides need not be limited to those molecules containing only RNA, but further encompasses chemically modified nucleotides and non-nucleotides. In certain embodiments, the short interfering nucleic acid molecules lack 2′-hydroxy (2′-OH) containing nucleotides. In certain embodiments short interfering nucleic acids optionally do not include any ribonucleotides (e.g., nucleotides having a 2′-OH group). Such double-stranded oligonucleotides that do not require the presence of ribonucleotides within the molecule to support RNAi can however have an attached linker or linkers or other attached or associated groups, moieties, or chains containing one or more nucleotides with 2′-OH groups. Optionally, double-stranded oligonucleotides can comprise ribonucleotides at about 5, 10, 20, 30, 40, or 50% of the nucleotide positions. As used herein, the term siRNA is meant to be equivalent to other terms used to describe nucleic acid molecules that are capable of mediating sequence specific RNAi, for example short interfering RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), short hairpin RNA (shRNA), short interfering oligonucleotide, short interfering nucleic acid, short interfering modified oligonucleotide, chemically modified siRNA, post-transcriptional gene silencing RNA (ptgsRNA), and others. In addition, as used herein, the term RNAi is meant to be equivalent to other terms used to describe sequence specific RNA interference, such as post transcriptional gene silencing, translational inhibition, or epigenetics. For example, double-stranded oligonucleotides can be used to epigenetically silence genes at both the post-transcriptional level and the pre-transcriptional level. In a non-limiting example, epigenetic regulation of gene expression by siRNA molecules of the invention can result from siRNA mediated modification of chromatin structure or methylation pattern to alter gene expression (see, for example, Verdel et al., 2004, Science, 303, 672-676; Pal-Bhadra et al., 2004, Science, 303, 669-672; Allshire, 2002, Science, 297, 1818-1819; Volpe et al., 2002, Science, 297, 1833-1837; Jenuwein, 2002, Science, 297, 2215-2218; and Hall et al., 2002, Science, 297, 2232-2237).

It is contemplated that compounds and compositions of several embodiments provided herein can target ANGPTL3 by a dsRNA-mediated gene silencing or RNAi mechanism, including, e.g., “hairpin” or stem-loop double-stranded RNA effector molecules in which a single RNA strand with self-complementary sequences is capable of assuming a double-stranded conformation, or duplex dsRNA effector molecules comprising two separate strands of RNA. In various embodiments, the dsRNA consists entirely of ribonucleotides or consists of a mixture of ribonucleotides and deoxynucleotides, such as the RNA/DNA hybrids disclosed, for example, by WO 00/63364, filed Apr. 19, 2000, or U.S. Ser. No. 60/130,377, filed Apr. 21, 1999. The dsRNA or dsRNA effector molecule may be a single molecule with a region of self-complementarity such that nucleotides in one segment of the molecule base pair with nucleotides in another segment of the molecule. In various embodiments, a dsRNA that consists of a single molecule consists entirely of ribonucleotides or includes a region of ribonucleotides that is complementary to a region of deoxyribonucleotides. Alternatively, the dsRNA may include two different strands that have a region of complementarity to each other.

In various embodiments, both strands consist entirely of ribonucleotides, one strand consists entirely of ribonucleotides and one strand consists entirely of deoxyribonucleotides, or one or both strands contain a mixture of ribonucleotides and deoxyribonucleotides. In certain embodiments, the regions of complementarity are at least 70, 80, 90, 95, 98, or 100% complementary to each other and to a target nucleic acid sequence. In certain embodiments, the region of the dsRNA that is present in a double-stranded conformation includes at least 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 50, 75, 100, 200, 500, 1000, 2000 or 5000 nucleotides or includes all of the nucleotides in a cDNA or other target nucleic acid sequence being represented in the dsRNA.

In some embodiments, the dsRNA does not contain any single stranded regions, such as single stranded ends, or the dsRNA is a hairpin. In other embodiments, the dsRNA has one or more single stranded regions or overhangs. In certain embodiments, RNA/DNA hybrids include a DNA strand or region that is an antisense strand or region (e.g, has at least 70, 80, 90, 95, 98, or 100% complementarity to a target nucleic acid) and an RNA strand or region that is a sense strand or region (e.g, has at least 70, 80, 90, 95, 98, or 100% identity to a target nucleic acid), and vice versa.

In various embodiments, the RNA/DNA hybrid is made in vitro using enzymatic or chemical synthetic methods such as those described herein or those described in WO 00/63364, filed Apr. 19, 2000, or U.S. Ser. No. 60/130,377, filed Apr. 21, 1999. In other embodiments, a DNA strand synthesized in vitro is complexed with an RNA strand made in vivo or in vitro before, after, or concurrent with the transformation of the DNA strand into the cell. In yet other embodiments, the dsRNA is a single circular nucleic acid containing a sense and an antisense region, or the dsRNA includes a circular nucleic acid and either a second circular nucleic acid or a linear nucleic acid (see, for example, WO 00/63364, filed Apr. 19, 2000, or U.S. Ser. No. 60/130,377, filed Apr. 21, 1999.) Exemplary circular nucleic acids include lariat structures in which the free 5′ phosphoryl group of a nucleotide becomes linked to the 2′ hydroxyl group of another nucleotide in a loop back fashion.

In other embodiments, the dsRNA includes one or more modified nucleotides in which the 2′ position in the sugar contains a halogen (such as fluorine group) or contains an alkoxy group (such as a methoxy group) which increases the half-life of the dsRNA in vitro or in vivo compared to the corresponding dsRNA in which the corresponding 2′ position contains a hydrogen or an hydroxyl group. In yet other embodiments, the dsRNA includes one or more linkages between adjacent nucleotides other than a naturally-occurring phosphodiester linkage. Examples of such linkages include phosphoramide, phosphorothioate, and phosphorodithioate linkages. The dsRNAs may also be chemically modified nucleic acid molecules as taught in U.S. Pat. No. 6,673,661. In other embodiments, the dsRNA contains one or two capped strands, as disclosed, for example, by WO 00/63364, filed Apr. 19, 2000, or U.S. Ser. No. 60/130,377, filed Apr. 21, 1999.

In other embodiments, the dsRNA can be any of the at least partially dsRNA molecules disclosed in WO 00/63364, as well as any of the dsRNA molecules described in U.S. Provisional Application 60/399,998; and U.S. Provisional Application 60/419,532, and PCT/US2003/033466, the teaching of which is hereby incorporated by reference. Any of the dsRNAs may be expressed in vitro or in vivo using the methods described herein or standard methods, such as those described in WO 00/63364.

Occupancy

In certain embodiments, antisense compounds are not expected to result in cleavage or the target nucleic acid via RNase H or to result in cleavage or sequestration through the RISC pathway. In certain such embodiments, antisense activity may result from occupancy, wherein the presence of the hybridized antisense compound disrupts the activity of the target nucleic acid. In certain such embodiments, the antisense compound may be uniformly modified or may comprise a mix of modifications and/or modified and unmodified nucleosides.

Target Nucleic Acids, Target Regions and Nucleotide Sequences

Nucleotide sequences that encode ANGPTL3 include, without limitation, the following: the human sequence as set forth in GenBank Accession No. NM_014495.2 (incorporated herein as SEQ ID NO: 1) or GenBank Accession No. NT_032977.9 nucleotides 33032001 to 33046000 (incorporated herein as SEQ ID NO: 2). It is understood that the sequence set forth in each SEQ ID NO in the Examples contained herein is independent of any modification to a sugar moiety, an internucleoside linkage, or a nucleobase. As such, antisense compounds defined by a SEQ ID NO can comprise, independently, one or more modifications to a sugar moiety, an internucleoside linkage, or a nucleobase. Antisense compounds described by Isis Number (Isis No) indicate a combination ofnucleobase sequence and motif.

In certain embodiments, a target region is a structurally defined region of the target nucleic acid. For example, a target region can encompass a 3′ UTR, a 5′ UTR, an exon, an intron, an exon/intron junction, a coding region, a translation initiation region, translation termination region, or other defined nucleic acid region. The structurally defined regions for ANGPTL3 can be obtained by accession number from sequence databases such as NCBI and such information is incorporated herein by reference. In certain embodiments, a target region can encompass the sequence from a 5′ target site of one target segment within the target region to a 3′ target site of another target segment within the target region.

In certain embodiments, a “target segment” is a smaller, sub-portion of a target region within a nucleic acid. For example, a target segment can be the sequence of nucleotides of a target nucleic acid to which one or more antisense compound is targeted. “5′ target site” or “5′ start stie” refers to the 5′-most nucleotide of a target segment. “3′ target site” or “3′ stop site” refers to the 3′-most nucleotide of a target segment.

Targeting includes determination of at least one target segment to which an antisense compound hybridizes, such that a desired effect occurs. In certain embodiments, the desired effect is a reduction in mRNA target nucleic acid levels. In certain embodiments, the desired effect is reduction of levels of protein encoded by the target nucleic acid or a phenotypic change associated with the target nucleic acid.

A target region can contain one or more target segments. Multiple target segments within a target region can be overlapping. Alternatively, they can be non-overlapping. In certain embodiments, target segments within a target region are separated by no more than about 300 nucleotides. In certain embodiments, target segments within a target region are separated by a number of nucleotides that is, is about, is no more than, is no more than about, 250, 200, 150, 100, 90, 80, 70, 60, 50, 40, 30, 20, or 10 nucleotides on the target nucleic acid, or is a range defined by any two of the preceding values. In certain embodiments, target segments within a target region are separated by no more than, or no more than about, 5 nucleotides on the target nucleic acid. In certain embodiments, target segments are contiguous. Contemplated are target regions defined by a range having a starting nucleic acid that is any of the 5′ target sites or 3′ target sites listed herein.

Suitable target segments can be found within a 5′ UTR, a coding region, a 3′ UTR, an intron, an exon, or an exon/intron junction. Target segments containing a start codon or a stop codon are also suitable target segments. A suitable target segment can specifically exclude a certain structurally defined region such as the start codon or stop codon.

The determination of suitable target segments can include a comparison of the sequence of a target nucleic acid to other sequences throughout the genome. For example, the BLAST algorithm can be used to identify regions of similarity amongst different nucleic acids. This comparison can prevent the selection of antisense compound sequences that can hybridize in a non-specific manner to sequences other than a selected target nucleic acid (i.e., non-target or off-target sequences).

There can be variation in activity (e.g., as defined by percent reduction of target nucleic acid levels) of the antisense compounds within an active target region. In certain embodiments, reductions in ANGPTL3 mRNA levels are indicative of inhibition of ANGPTL3 protein expression. Reductions in levels of an ANGPTL3 protein are also indicative of inhibition of target mRNA expression. Further, phenotypic changes, such as a reduction of the level of cholesterol, LDL, triglyceride, or glucose, can be indicative of inhibition of ANGPTL3 mRNA and/or protein expression.

Hybridization

In some embodiments, hybridization occurs between an antisense compound disclosed herein and an ANGPTL3 nucleic acid. The most common mechanism of hybridization involves hydrogen bonding (e.g., Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding) between complementary nucleobases of the nucleic acid molecules.

Hybridization can occur under varying conditions. Stringent conditions are sequence-dependent and are determined by the nature and composition of the nucleic acid molecules to be hybridized.

Methods of determining whether a sequence is specifically hybridizable to a target nucleic acid are well known in the art (Sambrook and Russell, Molecular Cloning: A Laboratory Manual, 3^(rd) Ed., 2001). In certain embodiments, the antisense compounds provided herein are specifically hybridizable with an ANGPTL3 nucleic acid.

Complementarity

An antisense compound and a target nucleic acid are complementary to each other when a sufficient number of nucleobases of the antisense compound can hydrogen bond with the corresponding nucleobases of the target nucleic acid, such that a desired effect will occur (e.g., antisense inhibition of a target nucleic acid, such as an ANGPTL3 nucleic acid).

An antisense compound can hybridize over one or more segments of an ANGPTL3 nucleic acid such that intervening or adjacent segments are not involved in the hybridization event (e.g., a loop structure, mismatch or hairpin structure).

In certain embodiments, the antisense compounds provided herein, or a specified portion thereof, are, or are at least, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% complementary to an ANGPTL3 nucleic acid, a target region, target segment, or specified portion thereof. In certain embodiments, the antisense compounds provided herein, or a specified portion thereof, are, or are at least, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% complementary to the sequence of one or more of SEQ ID NOs: 1-2. Percent complementarity of an antisense compound with a target nucleic acid can be determined using routine methods.

For example, an antisense compound in which 18 of 20 nucleobases of the antisense compound are complementary to a target region, and would therefore specifically hybridize, would represent 90 percent complementarity. In this example, the remaining noncomplementary nucleobases can be clustered or interspersed with complementary nucleobases and need not be contiguous to each other or to complementary nucleobases. As such, an antisense compound which is 18 nucleobases in length having 4 (four) noncomplementary nucleobases which are flanked by two regions of complete complementarity with the target nucleic acid would have 77.8% overall complementarity with the target nucleic acid and would thus fall within the scope of the present invention. Percent complementarity of an antisense compound with a region of a target nucleic acid can be determined routinely using BLAST programs (basic local alignment search tools) and PowerBLAST programs known in the art (Altschul et al., J. Mol. Biol., 1990, 215, 403 410; Zhang and Madden, Genome Res., 1997, 7, 649 656). Percent homology, sequence identity or complementarity, can be determined by, for example, the Gap program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, Madison Wis.), using default settings, which uses the algorithm of Smith and Waterman (Adv. Appl. Math., 1981, 2, 482 489).

In certain embodiments, the antisense compounds provided herein, or specified portions thereof, are fully complementary (i.e. 100% complementary) to a target nucleic acid, or specified portion thereof. For example, an antisense compound can be fully complementary to an ANGPTL3 nucleic acid, or a target region, or a target segment or target sequence thereof. As used herein, “fully complementary” means each nucleobase of an antisense compound is capable of precise base pairing with the corresponding nucleobases of a target nucleic acid. For example, a 20 nucleobase antisense compound is fully complementary to a target sequence that is 400 nucleobases long, so long as there is a corresponding 20 nucleobase portion of the target nucleic acid that is fully complementary to the antisense compound. Fully complementary can also be used in reference to a specified portion of the first and/or the second nucleic acid. For example, a 20 nucleobase portion of a 30 nucleobase antisense compound can be “fully complementary” to a target sequence that is 400 nucleobases long. The 20 nucleobase portion of the 30 nucleobase oligonucleotide is fully complementary to the target sequence if the target sequence has a corresponding 20 nucleobase portion wherein each nucleobase is complementary to the 20 nucleobase portion of the antisense compound. At the same time, the entire 30 nucleobase antisense compound can be fully complementary to the target sequence, depending on whether the remaining 10 nucleobases of the antisense compound are also complementary to the target sequence.

The location of a non-complementary nucleobase can be at the 5′ end or 3′ end of the antisense compound. Alternatively, the non-complementary nucleobase or nucleobases can be at an internal position of the antisense compound. When two or more non-complementary nucleobases are present, they can be either contiguous (i.e. linked) or non-contiguous. In one embodiment, a non-complementary nucleobase is located in the wing segment of a gapmer antisense oligonucleotide.

In certain embodiments, antisense compounds that are, or are up to 10, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleobases in length comprise no more than 4, no more than 3, no more than 2, or no more than 1 non-complementary nucleobase(s) relative to a target nucleic acid, such as an ANGPTL3 nucleic acid, or specified portion thereof.

In certain embodiments, antisense compounds that are, or are up to 10, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleobases in length comprise no more than 6, no more than 5, no more than 4, no more than 3, no more than 2, or no more than 1 non-complementary nucleobase(s) relative to a target nucleic acid, such as an ANGPTL3 nucleic acid, or specified portion thereof.

The antisense compounds provided herein also include those which are complementary to a portion of a target nucleic acid. As used herein, “portion” refers to a defined number of contiguous (i.e. linked) nucleobases within a region or segment of a target nucleic acid. A “portion” can also refer to a defined number of contiguous nucleobases of an antisense compound. In certain embodiments, the antisense compounds, are complementary to at least an 8 nucleobase portion of a target segment. In certain embodiments, the antisense compounds are complementary to at least a 10 nucleobase portion of a target segment. In certain embodiments, the antisense compounds are complementary to at least a 15 nucleobase portion of a target segment. Also contemplated are antisense compounds that are complementary to at least an 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more nucleobase portion of a target segment, or a range defined by any two of these values.

Identity

The antisense compounds provided herein can also have a defined percent identity to a particular nucleotide sequence, SEQ ID NO, or the sequence of a compound represented by a specific Isis number, or portion thereof. As used herein, an antisense compound is identical to the sequence disclosed herein if it has the same nucleobase pairing ability. For example, a RNA which contains uracil in place of thymidine in a disclosed DNA sequence would be considered identical to the DNA sequence since both uracil and thymidine pair with adenine. Shortened and lengthened versions of the antisense compounds described herein as well as compounds having non-identical bases relative to the antisense compounds provided herein also are contemplated. The non-identical bases can be adjacent to each other or dispersed throughout the antisense compound. Percent identity of an antisense compound is calculated according to the number of bases that have identical base pairing relative to the sequence to which it is being compared.

In certain embodiments, the antisense compounds, or portions thereof, are at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to one or more of the antisense compounds or SEQ ID NOs, or a portion thereof, disclosed herein.

Modifications

A nucleoside is a base-sugar combination. The nucleobase (also known as base) portion of the nucleoside is normally a heterocyclic base moiety. Nucleotides are nucleosides that further include a phosphate group covalently linked to the sugar portion of the nucleoside. For those nucleosides that include a pentofuranosyl sugar, the phosphate group can be linked to the 2′, 3′ or 5′ hydroxyl moiety of the sugar. Oligonucleotides are formed through the covalent linkage of adjacent nucleosides to one another, to form a linear polymeric oligonucleotide. Within the oligonucleotide structure, the phosphate groups are commonly referred to as forming the internucleoside linkages of the oligonucleotide.

Modifications to antisense compounds encompass substitutions or changes to internucleoside linkages, sugar moieties, or nucleobases. Modified antisense compounds are often preferred over native forms because of desirable properties such as, for example, enhanced cellular uptake, enhanced affinity for nucleic acid target, increased stability in the presence of nucleases, or increased inhibitory activity.

Chemically modified nucleosides can also be employed to increase the binding affinity of a shortened or truncated antisense oligonucleotide for its target nucleic acid. Consequently, comparable results can often be obtained with shorter antisense compounds that have such chemically modified nucleosides.

Modified Internucleoside Linkages

The naturally occurring internucleoside linkage of RNA and DNA is a 3′ to 5′ phosphodiester linkage. Antisense compounds having one or more modified, i.e. non-naturally occurring, internucleoside linkages are often selected over antisense compounds having naturally occurring internucleoside linkages because of desirable properties such as, for example, enhanced cellular uptake, enhanced affinity for target nucleic acids, and increased stability in the presence of nucleases.

Oligonucleotides having modified internucleoside linkages include internucleoside linkages that retain a phosphorus atom as well as internucleoside linkages that do not have a phosphorus atom. Representative phosphorus containing internucleoside linkages include, but are not limited to, phosphodiesters, phosphotriesters, methylphosphonates, phosphoramidate, and phosphorothioates. Methods of preparation of phosphorous-containing and non-phosphorous-containing linkages are well known.

In certain embodiments, antisense compounds targeted to an ANGPTL3 nucleic acid comprise one or more modified internucleoside linkages. In certain embodiments, the modified internucleoside linkages are phosphorothioate linkages. In certain embodiments, each internucleoside linkage of an antisense compound is a phosphorothioate internucleoside linkage.

In certain embodiments, oligonucleotides comprise modified internucleoside linkages arranged along the oligonucleotide or region thereof in a defined pattern or modified internucleoside linkage motif. In certain embodiments, internucleoside linkages are arranged in a gapped motif. In such embodiments, the internucleoside linkages in each of two wing regions are different from the internucleoside linkages in the gap region. In certain embodiments the internucleoside linkages in the wings are phosphodiester and the internucleoside linkages in the gap are phosphorothioate. The nucleoside motif is independently selected, so such oligonucleotides having a gapped internucleoside linkage motif may or may not have a gapped nucleoside motif and if it does have a gapped nucleoside motif, the wing and gap lengths may or may not be the same.

In certain embodiments, oligonucleotides comprise a region having an alternating internucleoside linkage motif. In certain embodiments, oligonucleotides of the present invention comprise a region of uniformly modified internucleoside linkages. In certain such embodiments, the oligonucleotide comprises a region that is uniformly linked by phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide is uniformly linked by phosphorothioate. In certain embodiments, each internucleoside linkage of the oligonucleotide is selected from phosphodiester and phosphorothioate. In certain embodiments, each internucleoside linkage of the oligonucleotide is selected from phosphodiester and phosphorothioate and at least one internucleoside linkage is phosphorothioate.

In certain embodiments, the oligonucleotide comprises at least 6 phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least 8 phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least 10 phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least one block of at least 6 consecutive phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least one block of at least 8 consecutive phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least one block of at least 10 consecutive phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least block of at least one 12 consecutive phosphorothioate internucleoside linkages. In certain such embodiments, at least one such block is located at the 3′ end of the oligonucleotide. In certain such embodiments, at least one such block is located within 3 nucleosides of the 3′ end of the oligonucleotide.

In certain embodiments, oligonucleotides comprise one or more methylphosponate linkages. In certain embodiments, oligonucleotides having a gapmer nucleoside motif comprise a linkage motif comprising all phosphorothioate linkages except for one or two methylphosponate linkages. In certain embodiments, one methylphosponate linkage is in the central gap of an oligonucleotide having a gapmer nucleoside motif.

In certain embodiments, it is desirable to arrange the number of phosphorothioate internucleoside linkages and phosphodiester internucleoside linkages to maintain nuclease resistance. In certain embodiments, it is desirable to arrange the number and position of phosphorothioate internucleoside linkages and the number and position of phosphodiester internucleoside linkages to maintain nuclease resistance. In certain embodiments, the number of phosphorothioate internucleoside linkages may be decreased and the number of phosphodiester internucleoside linkages may be increased. In certain embodiments, the number of phosphorothioate internucleoside linkages may be decreased and the number of phosphodiester internucleoside linkages may be increased while still maintaining nuclease resistance. In certain embodiments it is desirable to decrease the number of phosphorothioate internucleoside linkages while retaining nuclease resistance. In certain embodiments it is desirable to increase the number of phosphodiester internucleoside linkages while retaining nuclease resistance.

Modified Sugar Moieties

Antisense compounds of the invention can optionally contain one or more nucleosides wherein the sugar group has been modified. Such sugar modified nucleosides may impart enhanced nuclease stability, increased binding affinity, or some other beneficial biological property to the antisense compounds. In certain embodiments, nucleosides comprise chemically modified ribofuranose ring moieties. Examples of chemically modified ribofuranose rings include without limitation, addition of substitutent groups (including 5′ and 2′ substituent groups, bridging of non-geminal ring atoms to form bicyclic nucleic acids (BNA), replacement of the ribosyl ring oxygen atom with S, N(R), or C(R₁)(R₂) (R, R₁ and R₂ are each independently H, C₁-C₁₂ alkyl or a protecting group) and combinations thereof. Examples of chemically modified sugars include 2′-F-5′-methyl substituted nucleoside (see PCT International Application WO 2008/101157 Published on Aug. 21, 2008 for other disclosed 5′,2′-bis substituted nucleosides) or replacement of the ribosyl ring oxygen atom with S with further substitution at the 2′-position (see published U.S. Patent Application US2005-0130923, published on Jun. 16, 2005) or alternatively 5′-substitution of a BNA (see PCT International Application WO 2007/134181 Published on Nov. 22, 2007 wherein LNA is substituted with for example a 5′-methyl or a 5′-vinyl group).

Examples of nucleosides having modified sugar moieties include without limitation nucleosides comprising 5′-vinyl, 5′-methyl (R or S), 4′-S, 2′-F, 2′-OCH₃, 2′-OCH₂CH₃, 2′-OCH₂CH₂F and 2′-O(CH₂)₂OCH₃ substituent groups. The substituent at the 2′ position can also be selected from allyl, amino, azido, thio, O-allyl, O—C₁-C₁₀ alkyl, OCF₃, OCH₂F, O(CH₂)₂SCH₃, O(CH₂)₂—O—N(R_(m))(R_(n)), O—CH₂—C(═O)—N(R_(m))(R_(n)), and O—CH₂—C(═O)—N(R₁)—(CH₂)₂—N(R_(m))(R_(n)), where each R_(l), R_(m) and R_(n) is, independently, H or substituted or unsubstituted C₁-C₁₀ alkyl.

As used herein, “bicyclic nucleosides” refer to modified nucleosides comprising a bicyclic sugar moiety. Examples of bicyclic nucleic acids (BNAs) include without limitation nucleosides comprising a bridge between the 4′ and the 2′ ribosyl ring atoms. In certain embodiments, antisense compounds provided herein include one or more BNA nucleosides wherein the bridge comprises one of the formulas: 4′-(CH₂)—O-2′ (LNA); 4′-(CH₂)—S-2′; 4′-(CH₂)₂—O-2′ (ENA); 4′-CH(CH₃)—O-2′ and 4′-CH(CH₂OCH₃)—O-2′ (and analogs thereof see U.S. Pat. No. 7,399,845, issued on Jul. 15, 2008); 4′-C(CH₃)(CH₃)—O-2′ (and analogs thereof see PCT/US2008/068922 published as WO 2009/006478, published Jan. 8, 2009); 4′-CH₂—N(OCH₃)-2′ (and analogs thereof see PCT/US2008/064591 published as WO/2008/150729, published Dec. 11, 2008); 4′-CH₂—O—N(CH₃)-2′ (see published U.S. Patent Application US2004-0171570, published Sep. 2, 2004); 4′-CH₂—N(R)—O-2′, wherein R is H, C₁-C₁₂ alkyl, or a protecting group (see U.S. Pat. No. 7,427,672, issued on Sep. 23, 2008); 4′-CH₂—C(H)(CH₃)-2′ (see Zhou et al., J. Org. Chem., 2009, 74, 118-134); and 4′-CH₂—C(═CH₂)-2′ (and analogs thereof see PCT/US2008/066154 published as WO 2008/154401, published on Dec. 8, 2008).

Further bicyclic nucleosides have been reported in published literature (see for example: Srivastava et al., J. Am. Chem. Soc., 2007, 129(26) 8362-8379; Frieden et al., Nucleic Acids Research, 2003, 21, 6365-6372; Elayadi et al., Curr. Opinion Invens. Drugs, 2001, 2, 558-561; Braasch et al., Chem. Biol., 2001, 8, 1-7; Orum et al., Curr. Opinion Mol. Ther., 2001, 3, 239-243; Wahlestedt et al., Proc. Natl. Acad. Sci. U.S.A, 2000, 97, 5633-5638; Singh et al., Chem. Commun., 1998, 4, 455-456; Koshkin et al., Tetrahedron, 1998, 54, 3607-3630; Kumar et al., Bioorg. Med. Chem. Lett., 1998, 8, 2219-2222; Singh et al., J. Org. Chem., 1998, 63, 10035-10039; U.S. Pat. Nos. 7,741,457; 7,399,845; 7,053,207; 7,034,133; 6,794,499; 6,770,748; 6,670,461; 6,525,191; 6,268,490; U.S. Patent Publication Nos.: US2008-0039618; US2007-0287831; US2004-0171570; U.S. Patent Application Ser. Nos. 61/097,787; 61/026,995; and International applications: WO 2009/006478; WO 2008/154401; WO 2008/150729; WO 2009/100320; WO 2011/017521; WO 2009/067647; WO 2010/036698; WO 2007/134181; WO 2005/021570; WO 2004/106356; WO 99/14226. Each of the foregoing bicyclic nucleosides can be prepared having one or more stereochemical sugar configurations including for example α-L-ribofuranose and β-D-ribofuranose (see PCT international application PCT/DK98/00393, published on Mar. 25, 1999 as WO 99/14226).

As used herein, “monocylic nucleosides” refer to nucleosides comprising modified sugar moieties that are not bicyclic sugar moieties. In certain embodiments, the sugar moiety, or sugar moiety analogue, of a nucleoside may be modified or substituted at any position.

As used herein, “4′-2′ bicyclic nucleoside” or “4′ to 2′ bicyclic nucleoside” refers to a bicyclic nucleoside comprising a furanose ring comprising a bridge connecting two carbon atoms of the furanose ring connects the 2′ carbon atom and the 4′ carbon atom of the sugar ring.

In certain embodiments, bicyclic sugar moieties of BNA nucleosides include, but are not limited to, compounds having at least one bridge between the 4′ and the 2′ carbon atoms of the pentofuranosyl sugar moiety including without limitation, bridges comprising 1 or from 1 to 4 linked groups independently selected from —[C(R_(a))(R_(b))]_(n)—, —C(R_(a))═C(R_(b))—, —C(R_(a))═N—, —C(═NR_(a))—, —C(═O)—, —C(═S)—, —O—, —Si(R_(a))₂—, —S(═O)_(x)—, and —N(R_(a))—; wherein: x is 0, 1, or 2; n is 1, 2, 3, or 4; each R_(a) and R_(b) is, independently, H, a protecting group, hydroxyl, C₁-C₁₂ alkyl, substituted C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, substituted C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl, substituted C₂-C₁₂ alkynyl, C₅-C₂₀ aryl, substituted C₅-C₂₀ aryl, heterocycle radical, substituted heterocycle radical, heteroaryl, substituted heteroaryl, C₅-C₇ alicyclic radical, substituted C₅-C₇ alicyclic radical, halogen, OJ₁, NJ₁J₂, SJ₁, N₃, COOJ₁, acyl (C(═O)—H), substituted acyl, CN, sulfonyl (S(═O)₂-J₁), or sulfoxyl (S(═O)-J₁); and each J₁ and J₂ is, independently, H, C₁-C₁₂ alkyl, substituted C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, substituted C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl, substituted C₂-C₁₂ alkynyl, C₅-C₂₀ aryl, substituted C₅-C₂₀ aryl, acyl (C(═O)—H), substituted acyl, a heterocycle radical, a substituted heterocycle radical, C₁-C₁₂ aminoalkyl, substituted C₁-C₁₂ aminoalkyl or a protecting group.

In certain embodiments, the bridge of a bicyclic sugar moiety is, —[C(R_(a))(R_(b))]_(n)—, —[C(R_(a))(R_(b))]_(n)—O—, —C(R_(a)R_(b))—N(R)—O— or —C(R_(a)R_(b))—O—N(R)—. In certain embodiments, the bridge is 4′-CH₂-2′, 4′-(CH₂)₂-2′, 4′-(CH₂)₃-2′, 4′-CH₂—O-2′, 4′-(CH₂)₂—O-2′, 4′-CH₂—O—N(R)-2′ and 4′-CH₂—N(R)—O-2′- wherein each R is, independently, H, a protecting group or C₁-C₁₂ alkyl.

In certain embodiments, bicyclic nucleosides are further defined by isomeric configuration. For example, a nucleoside comprising a 4′-(CH₂)—O-2′ bridge, may be in the α-L configuration or in the β-D configuration. Previously, α-L-methyleneoxy (4′-CH₂—O-2′) BNA's have been incorporated into antisense oligonucleotides that showed antisense activity (Frieden et al., Nucleic Acids Research, 2003, 21, 6365-6372).

In certain embodiments, bicyclic nucleosides include those having a 4′ to 2′ bridge wherein such bridges include without limitation, α-L-4′-(CH₂)—O-2′, 3-D-4′-CH₂—O-2′, 4′-(CH₂)₂—O-2′, 4′-CH₂—O—N(R)-2′, 4′-CH₂—N(R)—O-2′, 4′-CH(CH₃)—O-2′, 4′-CH₂—S-2′, 4′-CH₂—N(R)-2′, 4′-CH₂—CH(CH₃)-2′, and 4′-(CH₂)₃-2′, wherein R is H, a protecting group or C₁-C₁₂ alkyl.

In certain embodiment, bicyclic nucleosides have the formula:

wherein:

Bx is a heterocyclic base moiety;

-Q_(a)-Q_(b)-Q_(c)- is —CH₂—N(R_(c))—CH₂—, —C(═O)—N(R_(c))—CH₂—, —CH₂—O—N(R_(c))—, —CH₂—N(R_(c))—O— or —N(R_(c))—O—CH₂;

R_(c) is C₁-C₁₂ alkyl or an amino protecting group; and

T_(a) and T_(b) are each, independently H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety or a covalent attachment to a support medium.

In certain embodiments, bicyclic nucleosides have the formula:

wherein:

Bx is a heterocyclic base moiety;

T_(a) and T_(b) are each, independently H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety or a covalent attachment to a support medium;

Z_(a) is C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, substituted C₁-C₆ alkyl, substituted C₂-C₆ alkenyl, substituted C₂-C₆ alkynyl, acyl, substituted acyl, substituted amide, thiol or substituted thiol.

In one embodiment, each of the substituted groups, is, independently, mono or poly substituted with substituent groups independently selected from halogen, oxo, hydroxyl, OJ_(c), NJ_(c)J_(d), SJ_(c), N₃, OC(═X)J_(c), and NJ_(e)C(═X)NJ_(c)J_(d), wherein each J_(c), J_(d) and J_(e) is, independently, H, C₁-C₆ alkyl, or substituted C₁-C₆ alkyl and X is O or NJ_(c).

In certain embodiments, bicyclic nucleosides have the formula:

wherein:

Bx is a heterocyclic base moiety;

T_(a) and T_(b) are each, independently H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety or a covalent attachment to a support medium;

Z_(b) is C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, substituted C₁-C₆ alkyl, substituted C₂-C₆ alkenyl, substituted C₂-C₆ alkynyl or substituted acyl (C(═O)—).

In certain embodiments, bicyclic nucleosides have the formula:

wherein:

Bx is a heterocyclic base moiety;

T_(a) and T_(b) are each, independently H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety or a covalent attachment to a support medium;

R_(d) is C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl or substituted C₂-C₆ alkynyl;

each q_(a), q_(b), q_(c) and q_(d) is, independently, H, halogen, C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl or substituted C₂-C₆ alkynyl, C₁-C₆ alkoxyl, substituted C₁-C₆ alkoxyl, acyl, substituted acyl, C₁-C₆ aminoalkyl or substituted C₁-C₆ aminoalkyl;

In certain embodiments, bicyclic nucleosides have the formula:

wherein:

Bx is a heterocyclic base moiety;

T_(a) and T_(b) are each, independently H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety or a covalent attachment to a support medium; q_(a), q_(b), q_(e) and q_(f) are each, independently, hydrogen, halogen, C₁-C₁₂ alkyl, substituted C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, substituted C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl, substituted C₂-C₁₂ alkynyl, C₁-C₁₂ alkoxy, substituted C₁-C₁₂ alkoxy, OJ_(j), SJ_(j), SOJ_(j), SO₂J_(j), NJ_(j)J_(k), N₃, CN, C(═O)OJ_(j), C(═O)N_(j)J_(k), C(═O)J_(j), O—C(═O)NJ_(j)J_(k), N(H)C(═NH)NJ_(j)J_(k), N(H)C(═O)NJ_(j)J_(k) or N(H)C(═S)NJ_(j)J_(k);

or q_(e) and q_(f) together are ═C(q_(g))(q_(h));

q_(g) and q_(h) are each, independently, H, halogen, C₁-C₁₂ alkyl or substituted C₁-C₁₂ alkyl.

The synthesis and preparation of adenine, cytosine, guanine, 5-methyl-cytosine, thymine and uracil bicyclic nucleosides having a 4′-CH₂—O-2′ bridge, along with their oligomerization, and nucleic acid recognition properties have been described (Koshkin et al., Tetrahedron, 1998, 54, 3607-3630). The synthesis of bicyclic nucleosides has also been described in WO 98/39352 and WO 99/14226.

Analogs of various bicyclic nucleosides that have 4′ to 2′ bridging groups such as 4′-CH₂—O-2′ and 4′-CH₂—S-2′, have also been prepared (Kumar et al., Bioorg. Med. Chem. Lett., 1998, 8, 2219-2222).

Preparation of oligodeoxyribonucleotide duplexes comprising bicyclic nucleosides for use as substrates for nucleic acid polymerases has also been described (Wengel et al., WO 99/14226). Furthermore, synthesis of 2′-amino-BNA, a novel conformationally restricted high-affinity oligonucleotide analog has been described in the art (Singh et al., J. Org. Chem., 1998, 63, 10035-10039). In addition, 2′-amino- and 2′-methylamino-BNA's have been prepared and the thermal stability of their duplexes with complementary RNA and DNA strands has been previously reported.

In certain embodiments, bicyclic nucleosides have the formula:

wherein:

Bx is a heterocyclic base moiety;

T_(a) and T_(b) are each, independently H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety or a covalent attachment to a support medium;

each q_(i), q_(j), q_(k) and q_(l) is, independently, H, halogen, C₁-C₁₂ alkyl, substituted C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, substituted C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl, substituted C₂-C₁₂ alkynyl, C₁-C₁₂ alkoxyl, substituted C₁-C₁₂ alkoxyl, OJ_(j), SJ_(j), SOJ_(j), SO₂J_(j), NJ_(j)J_(k), N₃, CN, C(═O)OJ_(j), C(═O)NJ_(j)J_(k), C(═O)J_(j), O—C(═O)NJ_(j)J_(k), N(H)C(═NH)NJ_(j)J_(k), N(H)C(═O)NJ_(j)J_(k) or N(H)C(═S)NJ_(j)J_(k); and

q_(i) and q_(j) or q_(i) and q_(k) together are ═C(q_(g))(q_(h)), wherein q_(g) and q_(h) are each, independently, H, halogen, C₁-C₁₂ alkyl or substituted C₁-C₁₂ alkyl.

One carbocyclic bicyclic nucleoside having a 4′-(CH₂)₃-2′ bridge and the alkenyl analog bridge 4′-CH═CH—CH₂-2′ have been described (Frier et al., Nucleic Acids Research, 1997, 25(22), 4429-4443 and Albaek et al., J. Org. Chem., 2006, 71, 7731-7740). The synthesis and preparation of carbocyclic bicyclic nucleosides along with their oligomerization and biochemical studies have also been described (Srivastava et al., J. Am. Chem. Soc. 2007, 129(26), 8362-8379).

In certain embodiments, bicyclic nucleosides include, but are not limited to, (A) α-L-methyleneoxy (4′-CH₂—O-2′) BNA, (B) β-D-methyleneoxy (4′-CH₂—O-2′) BNA, (C) ethyleneoxy (4′-(CH₂)₂—O-2′) BNA, (D) aminooxy (4′-CH₂—O—N(R)-2′) BNA, (E) oxyamino (4′-CH₂—N(R)—O-2′) BNA, (F) methyl(methyleneoxy) (4′-CH(CH₃)—O-2′) BNA (also referred to as constrained ethyl or cEt), (G) methylene-thio (4′-CH₂—S-2′) BNA, (H) methylene-amino (4′-CH₂—N(R)-2′) BNA, (I) methyl carbocyclic (4′-CH₂—CH(CH₃)-2′) BNA, (J) propylene carbocyclic (4′-(CH₂)₃-2′) BNA, and (K) vinyl BNA as depicted below.

wherein Bx is the base moiety and R is, independently, H, a protecting group, C₁-C₆ alkyl or C₁-C₆ alkoxy.

As used herein, the term “modified tetrahydropyran nucleoside” or “modified THP nucleoside” means a nucleoside having a six-membered tetrahydropyran “sugar” substituted for the pentofuranosyl residue in normal nucleosides and can be referred to as a sugar surrogate. Modified THP nucleosides include, but are not limited to, what is referred to in the art as hexitol nucleic acid (HNA), anitol nucleic acid (ANA), manitol nucleic acid (MNA) (see Leumann, Bioorg. Med. Chem., 2002, 10, 841-854) or fluoro HNA (F-HNA) having a tetrahydropyranyl ring system as illustrated below.

In certain embodiment, sugar surrogates are selected having the formula:

wherein:

Bx is a heterocyclic base moiety;

T₃ and T₄ are each, independently, an internucleoside linking group linking the tetrahydropyran nucleoside analog to the oligomeric compound or one of T₃ and T₄ is an internucleoside linking group linking the tetrahydropyran nucleoside analog to an oligomeric compound or oligonucleotide and the other of T₃ and T₄ is H, a hydroxyl protecting group, a linked conjugate group or a 5′ or 3′-terminal group;

q_(i), q₂, q₃, q₄, q₅, q₆ and q₇ are each independently, H, C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl or substituted C₂-C₆ alkynyl; and one of R₁ and R₂ is hydrogen and the other is selected from halogen, substituted or unsubstituted alkoxy, NJ₁J₂, SJ₁, N₃, OC(═X)J₁, OC(═X)NJ₁J₂, NJ₃C(═X)NJ₁J₂ and CN, wherein X is O, S or NJ₁ and each J₁, J₂ and J₃ is, independently, H or C₁-C₆ alkyl.

In certain embodiments, q_(i), q₂, q₃, q₄, q₅, q₆ and q₇ are each H. In certain embodiments, at least one of q_(i), q₂, q₃, q₄, q₅, q₆ and q₇ is other than H. In certain embodiments, at least one of q_(i), q₂, q₃, q₄, q₅, q₆ and q₇ is methyl. In certain embodiments, THP nucleosides are provided wherein one of R₁ and R₂ is F. In certain embodiments, R₁ is fluoro and R₂ is H; R₁ is methoxy and R₂ is H, and R₁ is methoxyethoxy and R₂ is H.

In certain embodiments, sugar surrogates comprise rings having more than 5 atoms and more than one heteroatom. For example nucleosides comprising morpholino sugar moieties and their use in oligomeric compounds has been reported (see for example: Braasch et al., Biochemistry, 2002, 41, 4503-4510; and U.S. Pat. Nos. 5,698,685; 5,166,315; 5,185,444; and 5,034,506). As used here, the term “morpholino” means a sugar surrogate having the following formula:

In certain embodiments, morpholinos may be modified, for example by adding or altering various substituent groups from the above morpholino structure. Such sugar surrogates are referred to herein as “modified morpholinos.”

Combinations of modifications are also provided without limitation, such as 2′-F-5′-methyl substituted nucleosides (see PCT International Application WO 2008/101157 published on Aug. 21, 2008 for other disclosed 5′,2′-bis substituted nucleosides) and replacement of the ribosyl ring oxygen atom with S and further substitution at the 2′-position (see published U.S. Patent Application US2005-0130923, published on Jun. 16, 2005) or alternatively 5′-substitution of a bicyclic nucleic acid (see PCT International Application WO 2007/134181, published on Nov. 22, 2007 wherein a 4′-CH₂—O-2′ bicyclic nucleoside is further substituted at the 5′ position with a 5′-methyl or a 5′-vinyl group). The synthesis and preparation of carbocyclic bicyclic nucleosides along with their oligomerization and biochemical studies have also been described (see, e.g., Srivastava et al., J. Am. Chem. Soc. 2007, 129(26), 8362-8379).

In certain embodiments, antisense compounds comprise one or more modified cyclohexenyl nucleosides, which is a nucleoside having a six-membered cyclohexenyl in place of the pentofuranosyl residue in naturally occurring nucleosides. Modified cyclohexenyl nucleosides include, but are not limited to those described in the art (see for example commonly owned, published PCT Application WO 2010/036696, published on Apr. 10, 2010, Robeyns et al., J. Am. Chem. Soc., 2008, 130(6), 1979-1984; Horvath et al., Tetrahedron Letters, 2007, 48, 3621-3623; Nauwelaerts et al., J. Am. Chem. Soc., 2007, 129(30), 9340-9348; Gu et al., Nucleosides, Nucleotides & Nucleic Acids, 2005, 24(5-7), 993-998; Nauwelaerts et al., Nucleic Acids Research, 2005, 33(8), 2452-2463; Robeyns et al., Acta Crystallographica, Section F: Structural Biology and Crystallization Communications, 2005, F61(6), 585-586; Gu et al., Tetrahedron, 2004, 60(9), 2111-2123; Gu et al., Oligonucleotides, 2003, 13(6), 479-489; Wang et al., J. Org. Chem., 2003, 68, 4499-4505; Verbeure et al., Nucleic Acids Research, 2001, 29(24), 4941-4947; Wang et al., J. Org. Chem., 2001, 66, 8478-82; Wang et al., Nucleosides, Nucleotides & Nucleic Acids, 2001, 20(4-7), 785-788; Wang et al., J. Am. Chem., 2000, 122, 8595-8602; Published PCT application, WO 06/047842; and Published PCT Application WO 01/049687; the text of each is incorporated by reference herein, in their entirety). Certain modified cyclohexenyl nucleosides have Formula X.

wherein independently for each of said at least one cyclohexenyl nucleoside analog of Formula X:

Bx is a heterocyclic base moiety;

T₃ and T₄ are each, independently, an internucleoside linking group linking the cyclohexenyl nucleoside analog to an antisense compound or one of T₃ and T₄ is an internucleoside linking group linking the tetrahydropyran nucleoside analog to an antisense compound and the other of T₃ and T₄ is H, a hydroxyl protecting group, a linked conjugate group, or a 5′- or 3′-terminal group; and

q₁, q₂, q₃, q₄, q₅, q₆, q₇, q₈ and q₉ are each, independently, H, C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl, substituted C₂-C₆ alkynyl or other sugar substituent group.

Many other monocyclic, bicyclic and tricyclic ring systems are known in the art and are suitable as sugar surrogates that can be used to modify nucleosides for incorporation into oligomeric compounds as provided herein (see for example review article: Leumann, Christian J. Bioorg. & Med. Chem., 2002, 10, 841-854). Such ring systems can undergo various additional substitutions to further enhance their activity.

As used herein, “2′-modified sugar” means a furanosyl sugar modified at the 2′ position. In certain embodiments, such modifications include substituents selected from: a halide, including, but not limited to substituted and unsubstituted alkoxy, substituted and unsubstituted thioalkyl, substituted and unsubstituted amino alkyl, substituted and unsubstituted alkyl, substituted and unsubstituted allyl, and substituted and unsubstituted alkynyl. In certain embodiments, 2′ modifications are selected from substituents including, but not limited to: O[(CH₂)_(n)O]_(m)CH₃, O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃, O(CH₂)_(n)F, O(CH₂)_(n)ONH₂, OCH₂C(═O)N(H)CH₃, and O(CH₂)_(n)ON[(CH₂)_(n)CH₃]₂, where n and m are from 1 to about 10. Other 2′-substituent groups can also be selected from: C₁-C₁₂ alkyl, substituted alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl, Br, CN, F, CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving pharmacokinetic properties, or a group for improving the pharmacodynamic properties of an antisense compound, and other substituents having similar properties. In certain embodiments, modified nucleosides comprise a 2′-MOE side chain (Baker et al., J. Biol. Chem., 1997, 272, 11944-12000). Such 2′-MOE substitution have been described as having improved binding affinity compared to unmodified nucleosides and to other modified nucleosides, such as 2′-O-methyl, O-propyl, and O-aminopropyl. Oligonucleotides having the 2′-MOE substituent also have been shown to be antisense inhibitors of gene expression with promising features for in vivo use (Martin, Helv. Chim. Acta, 1995, 78, 486-504; Altmann et al., Chimia, 1996, 50, 168-176; Altmann et al., Biochem. Soc. Trans., 1996, 24, 630-637; and Altmann et al., Nucleosides Nucleotides, 1997, 16, 917-926).

As used herein, “2′-modified” or “2′-substituted” refers to a nucleoside comprising a sugar comprising a substituent at the 2′ position other than H or OH. 2′-modified nucleosides, include, but are not limited to, bicyclic nucleosides wherein the bridge connecting two carbon atoms of the sugar ring connects the 2′ carbon and another carbon of the sugar ring; and nucleosides with non-bridging 2′ substituents, such as allyl, amino, azido, thio, O-allyl, O—C₁-C₁₀ alkyl, —OCF₃, O—(CH₂)₂—O—CH₃, 2′-O(CH₂)₂SCH₃, O—(CH₂)₂—O—N(R_(m))(R_(n)), or O—CH₂—C(═O)—N(R_(m))(R_(n)), where each R_(m) and R_(n) is, independently, H or substituted or unsubstituted C₁-C₁₀ alkyl. 2′-modified nucleosides may further comprise other modifications, for example at other positions of the sugar and/or at the nucleobase.

As used herein, “2′-F” refers to a nucleoside comprising a sugar comprising a fluoro group at the 2′ position of the sugar ring.

As used herein, “2′-OMe” or “2′-OCH₃”, “2′-O-methyl” or “2′-methoxy” each refers to a nucleoside comprising a sugar comprising an —OCH₃ group at the 2′ position of the sugar ring.

As used herein, “MOE” or “2′-MOE” or “2′-OCH₂CH₂OCH₃” or “2′-O-methoxyethyl” each refers to a nucleoside comprising a sugar comprising a —OCH₂CH₂OCH₃ group at the 2′ position of the sugar ring.

Methods for the preparations of modified sugars are well known to those skilled in the art. Some representative U.S. patents that teach the preparation of such modified sugars include without limitation, U.S.: 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,670,633; 5,700,920; 5,792,847 and 6,600,032 and International Application PCT/US2005/019219, filed Jun. 2, 2005 and published as WO 2005/121371 on Dec. 22, 2005, and each of which is herein incorporated by reference in its entirety.

As used herein, “oligonucleotide” refers to a compound comprising a plurality of linked nucleosides. In certain embodiments, one or more of the plurality of nucleosides is modified. In certain embodiments, an oligonucleotide comprises one or more ribonucleosides (RNA) and/or deoxyribonucleosides (DNA).

In nucleotides having modified sugar moieties, the nucleobase moieties (natural, modified or a combination thereof) are maintained for hybridization with an appropriate nucleic acid target.

In certain embodiments, antisense compounds comprise one or more nucleosides having modified sugar moieties. In certain embodiments, the modified sugar moiety is 2′-MOE. In certain embodiments, the 2′-MOE modified nucleosides are arranged in a gapmer motif. In certain embodiments, the modified sugar moiety is a bicyclic nucleoside having a (4′-CH(CH₃)—O-2′) bridging group. In certain embodiments, the (4′-CH(CH₃)—O-2′) modified nucleosides are arranged throughout the wings of a gapmer motif.

Modified Nucleobases

Nucleobase (or base) modifications or substitutions are structurally distinguishable from, yet functionally interchangeable with, naturally occurring or synthetic unmodified nucleobases. Both natural and modified nucleobases are capable of participating in hydrogen bonding. Such nucleobase modifications can impart nuclease stability, binding affinity or some other beneficial biological property to antisense compounds. Modified nucleobases include synthetic and natural nucleobases such as, for example, 5-methylcytosine (5-me-C). Certain nucleobase substitutions, including 5-methylcytosine substitutions, are particularly useful for increasing the binding affinity of an antisense compound for a target nucleic acid. For example, 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2° C. (Sanghvi, Y. S., Crooke, S. T. and Lebleu, B., eds., Antisense Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278).

Additional modified nucleobases include 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl (—C≡C—CH3) uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine.

Heterocyclic base moieties can also include those in which the purine or pyrimidine base is replaced with other heterocycles, for example 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone. Nucleobases that are particularly useful for increasing the binding affinity of antisense compounds include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2 aminopropyladenine, 5-propynyluracil and 5-propynylcytosine.

In certain embodiments, antisense compounds targeted to an ANGPTL3 nucleic acid comprise one or more modified nucleobases. In certain embodiments, shortened or gap-widened antisense oligonucleotides targeted to an ANGPTL3 nucleic acid comprise one or more modified nucleobases. In certain embodiments, the modified nucleobase is 5-methylcytosine. In certain embodiments, each cytosine is a 5-methylcytosine.

Compositions and Methods for Formulating Pharmaceutical Compositions

Antisense oligonucleotides can be admixed with pharmaceutically acceptable active or inert substance for the preparation of pharmaceutical compositions or formulations. Compositions and methods for the formulation of pharmaceutical compositions are dependent upon a number of criteria, including, but not limited to, route of administration, extent of disease, or dose to be administered.

Antisense compound targeted to an ANGPTL3 nucleic acid can be utilized in pharmaceutical compositions by combining the antisense compound with a suitable pharmaceutically acceptable diluent or carrier. A pharmaceutically acceptable diluent includes phosphate-buffered saline (PBS). PBS is a diluent suitable for use in compositions to be delivered parenterally. Accordingly, in one embodiment, employed in the methods described herein is a pharmaceutical composition comprising an antisense compound targeted to an ANGPTL3 nucleic acid and a pharmaceutically acceptable diluent. In certain embodiments, the pharmaceutically acceptable diluent is PBS. In certain embodiments, the antisense compound is an antisense oligonucleotide.

Pharmaceutical compositions comprising antisense compounds encompass any pharmaceutically acceptable salts, esters, or salts of such esters, or any other oligonucleotide which, upon administration to an animal, including a human, is capable of providing (directly or indirectly) the biologically active metabolite or residue thereof. Accordingly, for example, the disclosure is also drawn to pharmaceutically acceptable salts of antisense compounds, prodrugs, pharmaceutically acceptable salts of such prodrugs, and other bioequivalents. Suitable pharmaceutically acceptable salts include, but are not limited to, sodium and potassium salts.

A prodrug can include the incorporation of additional nucleosides at one or both ends of an antisense compound which are cleaved by endogenous nucleases within the body, to form the active antisense compound.

Conjugated Antisense Compounds

Antisense compounds can be covalently linked to one or more moieties or conjugates which enhance the activity, cellular distribution or cellular uptake of the resulting antisense oligonucleotides. Typical conjugate groups include cholesterol moieties and lipid moieties. Additional conjugate groups include carbohydrates, phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines, coumarins, and dyes.

Antisense compounds can also be modified to have one or more stabilizing groups that are generally attached to one or both termini of antisense compounds to enhance properties such as, for example, nuclease stability. Included in stabilizing groups are cap structures. These terminal modifications protect the antisense compound having terminal nucleic acids from exonuclease degradation, and can help in delivery and/or localization within a cell. The cap can be present at the 5′-terminus (5′-cap), or at the 3′-terminus (3′-cap), or can be present on both termini. Cap structures are well known in the art and include, for example, inverted deoxy abasic caps. Further 3′ and 5′-stabilizing groups that can be used to cap one or both ends of an antisense compound to impart nuclease stability include those disclosed in WO 03/004602 published on Jan. 16, 2003.

In certain embodiments, the present disclosure provides conjugated antisense compounds represented by the formula: A-B—C-D

E-F)_(q)

wherein

A is the antisense oligonucleotide;

B is the cleavable moiety

C is the conjugate linker

D is the branching group

each E is a tether;

each F is a ligand; and

q is an integer between 1 and 5.

In certain embodiments, conjugated antisense compounds are provided having the structure:

In certain embodiments, conjugated antisense compounds are provided having the structure:

In certain embodiments, conjugated antisense compounds are provided having the structure:

The present disclosure provides the following non-limiting numbered embodiments:

wherein:

-   -   T₂ is a nucleoside, a nucleotide, a monomeric subunit, or an         oligomeric compound.

In embodiments having more than one of a particular variable (e.g., more than one “m” or “n”), unless otherwise indicated, each such particular variable is selected independently. Thus, for a structure having more than one n, each n is selected independently, so they may or may not be the same as one another.

i. Certain Cleavable Moieties

In certain embodiments, a cleavable moiety is a cleavable bond. In certain embodiments, a cleavable moiety comprises a cleavable bond. In certain embodiments, the conjugate group comprises a cleavable moiety. In certain such embodiments, the cleavable moiety attaches to the antisense oligonucleotide. In certain such embodiments, the cleavable moiety attaches directly to the cell-targeting moiety. In certain such embodiments, the cleavable moiety attaches to the conjugate linker. In certain embodiments, the cleavable moiety comprises a phosphate or phosphodiester. In certain embodiments, the cleavable moiety is a cleavable nucleoside or nucleoside analog. In certain embodiments, the nucleoside or nucleoside analog comprises an optionally protected heterocyclic base selected from a purine, substituted purine, pyrimidine or substituted pyrimidine. In certain embodiments, the cleavable moiety is a nucleoside comprising an optionally protected heterocyclic base selected from uracil, thymine, cytosine, 4-N-benzoylcytosine, 5-methylcytosine, 4-N-benzoyl-5-methylcytosine, adenine, 6-N-benzoyladenine, guanine and 2-N-isobutyrylguanine. In certain embodiments, the cleavable moiety is 2′-deoxy nucleoside that is attached to the 3′ position of the antisense oligonucleotide by a phosphodiester linkage and is attached to the linker by a phosphodiester or phosphorothioate linkage. In certain embodiments, the cleavable moiety is 2′-deoxy adenosine that is attached to the 3′ position of the antisense oligonucleotide by a phosphodiester linkage and is attached to the linker by a phosphodiester or phosphorothioate linkage. In certain embodiments, the cleavable moiety is 2′-deoxy adenosine that is attached to the 3′ position of the antisense oligonucleotide by a phosphodiester linkage and is attached to the linker by a phosphodiester linkage.

In certain embodiments, the cleavable moiety is attached to the 3′ position of the antisense oligonucleotide. In certain embodiments, the cleavable moiety is attached to the 5′ position of the antisense oligonucleotide. In certain embodiments, the cleavable moiety is attached to a 2′ position of the antisense oligonucleotide. In certain embodiments, the cleavable moiety is attached to the antisense oligonucleotide by a phosphodiester linkage. In certain embodiments, the cleavable moiety is attached to the linker by either a phosphodiester or a phosphorothioate linkage. In certain embodiments, the cleavable moiety is attached to the linker by a phosphodiester linkage. In certain embodiments, the conjugate group does not include a cleavable moiety.

In certain embodiments, the cleavable moiety is cleaved after the complex has been administered to an animal only after being internalized by a targeted cell. Inside the cell the cleavable moiety is cleaved thereby releasing the active antisense oligonucleotide. While not wanting to be bound by theory it is believed that the cleavable moiety is cleaved by one or more nucleases within the cell. In certain embodiments, the one or more nucleases cleave the phosphodiester linkage between the cleavable moiety and the linker. In certain embodiments, the cleavable moiety has a structure selected from among the following:

wherein each of Bx, Bx₁, Bx₂, and Bx₃ is independently a heterocyclic base moiety. In certain embodiments, the cleavable moiety has a structure selected from among the following:

i. Certain Linkers

In certain embodiments, the conjugate groups comprise a linker. In certain such embodiments, the linker is covalently bound to the cleavable moiety. In certain such embodiments, the linker is covalently bound to the antisense oligonucleotide. In certain embodiments, the linker is covalently bound to a cell-targeting moiety. In certain embodiments, the linker further comprises a covalent attachment to a solid support. In certain embodiments, the linker further comprises a covalent attachment to a protein binding moiety. In certain embodiments, the linker further comprises a covalent attachment to a solid support and further comprises a covalent attachment to a protein binding moiety. In certain embodiments, the linker includes multiple positions for attachment of tethered ligands. In certain embodiments, the linker includes multiple positions for attachment of tethered ligands and is not attached to a branching group. In certain embodiments, the linker further comprises one or more cleavable bond. In certain embodiments, the conjugate group does not include a linker.

In certain embodiments, the linker includes at least a linear group comprising groups selected from alkyl, amide, disulfide, polyethylene glycol, ether, thioether (—S—) and hydroxylamino (—O—N(H)—) groups. In certain embodiments, the linear group comprises groups selected from alkyl, amide and ether groups. In certain embodiments, the linear group comprises groups selected from alkyl and ether groups. In certain embodiments, the linear group comprises at least one phosphorus linking group. In certain embodiments, the linear group comprises at least one phosphodiester group. In certain embodiments, the linear group includes at least one neutral linking group. In certain embodiments, the linear group is covalently attached to the cell-targeting moiety and the cleavable moiety. In certain embodiments, the linear group is covalently attached to the cell-targeting moiety and the antisense oligonucleotide. In certain embodiments, the linear group is covalently attached to the cell-targeting moiety, the cleavable moiety and a solid support. In certain embodiments, the linear group is covalently attached to the cell-targeting moiety, the cleavable moiety, a solid support and a protein binding moiety. In certain embodiments, the linear group includes one or more cleavable bond.

In certain embodiments, the linker includes the linear group covalently attached to a scaffold group. In certain embodiments, the scaffold includes a branched aliphatic group comprising groups selected from alkyl, amide, disulfide, polyethylene glycol, ether, thioether and hydroxylamino groups. In certain embodiments, the scaffold includes a branched aliphatic group comprising groups selected from alkyl, amide and ether groups. In certain embodiments, the scaffold includes at least one mono or polycyclic ring system. In certain embodiments, the scaffold includes at least two mono or polycyclic ring systems. In certain embodiments, the linear group is covalently attached to the scaffold group and the scaffold group is covalently attached to the cleavable moiety and the linker. In certain embodiments, the linear group is covalently attached to the scaffold group and the scaffold group is covalently attached to the cleavable moiety, the linker and a solid support. In certain embodiments, the linear group is covalently attached to the scaffold group and the scaffold group is covalently attached to the cleavable moiety, the linker and a protein binding moiety. In certain embodiments, the linear group is covalently attached to the scaffold group and the scaffold group is covalently attached to the cleavable moiety, the linker, a protein binding moiety and a solid support. In certain embodiments, the scaffold group includes one or more cleavable bond.

In certain embodiments, the linker includes a protein binding moiety. In certain embodiments, the protein binding moiety is a lipid such as for example including but not limited to cholesterol, cholic acid, adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone, 1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine), a vitamin (e.g., folate, vitamin A, vitamin E, biotin, pyridoxal), a peptide, a carbohydrate (e.g., monosaccharide, disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, polysaccharide), an endosomolytic component, a steroid (e.g., uvaol, hecigenin, diosgenin), a terpene (e.g., triterpene, e.g., sarsasapogenin, friedelin, epifriedelanol derivatized lithocholic acid), or a cationic lipid. In certain embodiments, the protein binding moiety is a C16 to C22 long chain saturated or unsaturated fatty acid, cholesterol, cholic acid, vitamin E, adamantane or 1-pentafluoropropyl.

In certain embodiments, a linker has a structure selected from among:

wherein each n is, independently, from 1 to 20; and p is from 1 to 6.

In certain embodiments, a linker has a structure selected from among:

wherein each n is, independently, from 1 to 20.

In certain embodiments, a linker has a structure selected from among:

wherein n is from 1 to 20.

In certain embodiments, a linker has a structure selected from among:

-   -   wherein each L is, independently, a phosphorus linking group or         a neutral linking group; and     -   each n is, independently, from 1 to 20.

In certain embodiments, a linker has a structure selected from among:

In certain embodiments, a linker has a structure selected from among:

In certain embodiments, a linker has a structure selected from among:

In certain embodiments, a linker has a structure selected from among:

wherein n is from 1 to 20.

In certain embodiments, a linker has a structure selected from among:

In certain embodiments, a linker has a structure selected from among:

In certain embodiments, a linker has a structure selected from among:

In certain embodiments, the conjugate linker has the structure:

In certain embodiments, the conjugate linker has the structure:

In certain embodiments, a linker has a structure selected from among:

In certain embodiments, a linker has a structure selected from among:

wherein each n is independently, 0, 1, 2, 3, 4, 5, 6, or 7.

ii. Certain Cell-Targeting Moieties

In certain embodiments, conjugate groups comprise cell-targeting moieties. Certain such cell-targeting moieties increase cellular uptake of antisense compounds. In certain embodiments, cell-targeting moieties comprise a branching group, one or more tether, and one or more ligand. In certain embodiments, cell-targeting moieties comprise a branching group, one or more tether, one or more ligand and one or more cleavable bond.

1. Certain Branching Groups

In certain embodiments, the conjugate groups comprise a targeting moiety comprising a branching group and at least two tethered ligands. In certain embodiments, the branching group attaches the conjugate linker. In certain embodiments, the branching group attaches the cleavable moiety. In certain embodiments, the branching group attaches the antisense oligonucleotide. In certain embodiments, the branching group is covalently attached to the linker and each of the tethered ligands. In certain embodiments, the branching group comprises a branched aliphatic group comprising groups selected from alkyl, amide, disulfide, polyethylene glycol, ether, thioether and hydroxylamino groups. In certain embodiments, the branching group comprises groups selected from alkyl, amide and ether groups. In certain embodiments, the branching group comprises groups selected from alkyl and ether groups. In certain embodiments, the branching group comprises a mono or polycyclic ring system. In certain embodiments, the branching group comprises one or more cleavable bond. In certain embodiments, the conjugate group does not include a branching group.

In certain embodiments, a branching group has a structure selected from among:

wherein each n is, independently, from 1 to 20;

j is from 1 to 3; and

m is from 2 to 6.

In certain embodiments, a branching group has a structure selected from among:

wherein each n is, independently, from 1 to 20; and

m is from 2 to 6.

In certain embodiments, a branching group has a structure selected from among:

In certain embodiments, a branching group has a structure selected from among:

wherein each A₁ is independently, O, S, C═O or NH; and

each n is, independently, from 1 to 20.

In certain embodiments, a branching group has a structure selected from among:

-   -   wherein each A₁ is independently, O, S, C═O or NH; and     -   each n is, independently, from 1 to 20.

In certain embodiments, a branching group has a structure selected from among:

wherein A₁ is O, S, C═O or NH; and

each n is, independently, from 1 to 20.

In certain embodiments, a branching group has a structure selected from among:

In certain embodiments, a branching group has a structure selected from among:

In certain embodiments, a branching group has a structure selected from among:

2. Certain Tethers

In certain embodiments, conjugate groups comprise one or more tethers covalently attached to the branching group. In certain embodiments, conjugate groups comprise one or more tethers covalently attached to the linking group. In certain embodiments, each tether is a linear aliphatic group comprising one or more groups selected from alkyl, ether, thioether, disulfide, amide and polyethylene glycol groups in any combination. In certain embodiments, each tether is a linear aliphatic group comprising one or more groups selected from alkyl, substituted alkyl, ether, thioether, disulfide, amide, phosphodiester and polyethylene glycol groups in any combination. In certain embodiments, each tether is a linear aliphatic group comprising one or more groups selected from alkyl, ether and amide groups in any combination. In certain embodiments, each tether is a linear aliphatic group comprising one or more groups selected from alkyl, substituted alkyl, phosphodiester, ether and amide groups in any combination. In certain embodiments, each tether is a linear aliphatic group comprising one or more groups selected from alkyl and phosphodiester in any combination. In certain embodiments, each tether comprises at least one phosphorus linking group or neutral linking group.

In certain embodiments, the tether includes one or more cleavable bond. In certain embodiments, the tether is attached to the branching group through either an amide or an ether group. In certain embodiments, the tether is attached to the branching group through a phosphodiester group. In certain embodiments, the tether is attached to the branching group through a phosphorus linking group or neutral linking group. In certain embodiments, the tether is attached to the branching group through an ether group. In certain embodiments, the tether is attached to the ligand through either an amide or an ether group. In certain embodiments, the tether is attached to the ligand through an ether group. In certain embodiments, the tether is attached to the ligand through either an amide or an ether group. In certain embodiments, the tether is attached to the ligand through an ether group.

In certain embodiments, each tether comprises from about 8 to about 20 atoms in chain length between the ligand and the branching group. In certain embodiments, each tether group comprises from about 10 to about 18 atoms in chain length between the ligand and the branching group. In certain embodiments, each tether group comprises about 13 atoms in chain length.

In certain embodiments, a tether has a structure selected from among:

wherein each n is, independently, from 1 to 20; and

each p is from 1 to about 6.

In certain embodiments, a tether has a structure selected from among:

In certain embodiments, a tether has a structure selected from among:

-   -   wherein each n is, independently, from 1 to 20.

In certain embodiments, a tether has a structure selected from among:

wherein L is either a phosphorus linking group or a neutral linking group;

Z₁ is C(═O)O—R₂;

Z₂ is H, C₁-C₆ alkyl or substituted C₁-C₆ alky;

R₂ is H, C₁-C₆ alkyl or substituted C₁-C₆ alky; and

each m₁ is, independently, from 0 to 20 wherein at least one m₁ is greater than 0 for

each tether.

In certain embodiments, a tether has a structure selected from among:

In certain embodiments, a tether has a structure selected from among:

-   -   wherein Z₂ is H or CH₃; and     -   each m₁ is, independently, from 0 to 20 wherein at least one m₁         is greater than 0 for     -   each tether.

In certain embodiments, a tether has a structure selected from among:

wherein each n is independently, 0, 1, 2, 3, 4, 5, 6, or 7.

In certain embodiments, a tether comprises a phosphorus linking group. In certain embodiments, a tether does not comprise any amide bonds. In certain embodiments, a tether comprises a phosphorus linking group and does not comprise any amide bonds.

3. Certain Ligands

In certain embodiments, the present disclosure provides ligands wherein each ligand is covalently attached to a tether. In certain embodiments, each ligand is selected to have an affinity for at least one type of receptor on a target cell. In certain embodiments, ligands are selected that have an affinity for at least one type of receptor on the surface of a mammalian liver cell. In certain embodiments, ligands are selected that have an affinity for the hepatic asialoglycoprotein receptor (ASGP-R). In certain embodiments, each ligand is a carbohydrate. In certain embodiments, each ligand is, independently selected from galactose, N-acetyl galactoseamine, mannose, glucose, glucosamone and fucose. In certain embodiments, each ligand is N-acetyl galactoseamine (GalNAc). In certain embodiments, the targeting moiety comprises 2 to 6 ligands. In certain embodiments, the targeting moiety comprises 3 ligands. In certain embodiments, the targeting moiety comprises 3 N-acetyl galactoseamine ligands.

In certain embodiments, the ligand is a carbohydrate, carbohydrate derivative, modified carbohydrate, multivalent carbohydrate cluster, polysaccharide, modified polysaccharide, or polysaccharide derivative. In certain embodiments, the ligand is an amino sugar or a thio sugar. For example, amino sugars may be selected from any number of compounds known in the art, for example glucosamine, sialic acid, α-D-galactosamine, N-Acetylgalactosamine, 2-acetamido-2-deoxy-D-galactopyranose (GalNAc), 2-Amino-3-O—[(R)-1-carboxyethyl]-2-deoxy-β-D-glucopyranose (β-muramic acid), 2-Deoxy-2-methylamino-L-glucopyranose, 4,6-Dideoxy-4-formamido-2,3-di-O-methyl-D-mannopyranose, 2-Deoxy-2-sulfoamino-D-glucopyranose and N-sulfo-D-glucosamine, and N-Glycoloyl-α-neuraminic acid. For example, thio sugars may be selected from the group consisting of 5-Thio-β-D-glucopyranose, Methyl 2,3,4-tri-O-acetyl-1-thio-6-O-trityl-α-D-glucopyranoside, 4-Thio-β-D-galactopyranose, and ethyl 3,4,6,7-tetra-O-acetyl-2-deoxy-1,5-dithio-α-D-gluco-heptopyranoside.

In certain embodiments, “GalNac” or “Gal-NAc” refers to 2-(Acetylamino)-2-deoxy-D-galactopyranose, commonly referred to in the literature as N-acetyl galactosamine. In certain embodiments, “N-acetyl galactosamine” refers to 2-(Acetylamino)-2-deoxy-D-galactopyranose. In certain embodiments, “GalNac” or “Gal-NAc” refers to 2-(Acetylamino)-2-deoxy-D-galactopyranose. In certain embodiments, “GalNac” or “Gal-NAc” refers to 2-(Acetylamino)-2-deoxy-D-galactopyranose, which includes both the β-form: 2-(Acetylamino)-2-deoxy-β-D-galactopyranose and α-form: 2-(Acetylamino)-2-deoxy-D-galactopyranose. In certain embodiments, both the β-form: 2-(Acetylamino)-2-deoxy-β-D-galactopyranose and α-form: 2-(Acetylamino)-2-deoxy-D-galactopyranose may be used interchangeably. Accordingly, in structures in which one form is depicted, these structures are intended to include the other form as well. For example, where the structure for an α-form: 2-(Acetylamino)-2-deoxy-D-galactopyranose is shown, this structure is intended to include the other form as well. In certain embodiments. In certain preferred embodiments, the 0-form 2-(Acetylamino)-2-deoxy-D-galactopyranose is the preferred embodiment.

In certain embodiments one or more ligand has a structure selected from among:

wherein each R₁ is selected from OH and NHCOOH.

In certain embodiments one or more ligand has a structure selected from among:

In certain embodiments one or more ligand has a structure selected from among:

In certain embodiments one or more ligand has a structure selected from among:

iii. Certain Conjugates

In certain embodiments, conjugate groups comprise the structural features above. In certain such embodiments, conjugate groups comprise the following structure:

wherein each n is, independently, from 1 to 20.

In certain such embodiments, conjugate groups comprise the following structure:

In certain such embodiments, conjugate groups have the following structure:

wherein each n is, independently, from 1 to 20;

Z is H or a linked solid support;

Q is an antisense compound;

X is O or S; and

Bx is a heterocyclic base moiety.

In certain such embodiments, conjugate groups have the following structure:

In certain such embodiments, conjugate groups have the following structure:

In certain such embodiments, conjugate groups comprise the following structure:

In certain such embodiments, conjugate groups comprise the following structure:

In certain such embodiments, conjugate groups have the following structure:

In certain such embodiments, conjugate groups have the following structure:

In certain such embodiments, conjugate groups have the following structure:

In certain such embodiments, conjugate groups have the following structure:

In certain embodiments, conjugates do not comprise a pyrrolidine.

b. Certain Conjugated Antisense Compounds

In certain embodiments, the conjugates are bound to a nucleoside of the antisense oligonucleotide at the 2′, 3′, of 5′ position of the nucleoside. In certain embodiments, a conjugated antisense compound has the following structure: A-B—C-D

E-F)_(q)

wherein

A is the antisense oligonucleotide;

B is the cleavable moiety

C is the conjugate linker

D is the branching group

each E is a tether;

each F is a ligand; and

q is an integer between 1 and 5.

In certain embodiments, a conjugated antisense compound has the following structure: A-C-D

E-F)_(q)

wherein

A is the antisense oligonucleotide;

C is the conjugate linker

D is the branching group

each E is a tether;

each F is a ligand; and

q is an integer between 1 and 5.

In certain such embodiments, the conjugate linker comprises at least one cleavable bond.

In certain such embodiments, the branching group comprises at least one cleavable bond.

In certain embodiments each tether comprises at least one cleavable bond.

In certain embodiments, the conjugates are bound to a nucleoside of the antisense oligonucleotide at the 2′, 3′, of 5′ position of the nucleoside.

In certain embodiments, a conjugated antisense compound has the following structure: A-B—C

E-F)_(q)

wherein

A is the antisense oligonucleotide;

B is the cleavable moiety

C is the conjugate linker

each E is a tether;

each F is a ligand; and

q is an integer between 1 and 5.

In certain embodiments, the conjugates are bound to a nucleoside of the antisense oligonucleotide at the 2′, 3′, of 5′ position of the nucleoside. In certain embodiments, a conjugated antisense compound has the following structure: A-C

E-F)_(q)

wherein

A is the antisense oligonucleotide;

C is the conjugate linker

each E is a tether;

each F is a ligand; and

q is an integer between 1 and 5.

In certain embodiments, a conjugated antisense compound has the following structure: A-B-D

E-F)_(q)

wherein

A is the antisense oligonucleotide;

B is the cleavable moiety

D is the branching group

each E is a tether;

each F is a ligand; and

q is an integer between 1 and 5.

In certain embodiments, a conjugated antisense compound has the following structure: A-D

E-F)_(q)

wherein

A is the antisense oligonucleotide;

D is the branching group

each E is a tether;

each F is a ligand; and

q is an integer between 1 and 5.

In certain such embodiments, the conjugate linker comprises at least one cleavable bond.

In certain embodiments each tether comprises at least one cleavable bond.

In certain embodiments, a conjugated antisense compound has a structure selected from among the following:

In certain embodiments, a conjugated antisense compound has a structure selected from among the following:

In certain embodiments, a conjugated antisense compound has a structure selected from among the following:

In certain embodiments, the conjugated antisense compound has the following structure:

Representative United States patents, United States patent application publications, and international patent application publications that teach the preparation of certain of the above noted conjugates, conjugated antisense compounds, tethers, linkers, branching groups, ligands, cleavable moieties as well as other modifications include without limitation, U.S. Pat. Nos. 5,994,517, 6,300,319, 6,660,720, 6,906,182, 7,262,177, 7,491,805, 8,106,022, 7,723,509, US 2006/0148740, US 2011/0123520, WO 2013/033230 and WO 2012/037254, each of which is incorporated by reference herein in its entirety.

Representative publications that teach the preparation of certain of the above noted conjugates, conjugated antisense compounds, tethers, linkers, branching groups, ligands, cleavable moieties as well as other modifications include without limitation, BIESSEN et al., “The Cholesterol Derivative of a Triantennary Galactoside with High Affinity for the Hepatic Asialoglycoprotein Receptor: a Potent Cholesterol Lowering Agent” J. Med. Chem. (1995) 38:1846-1852, BIESSEN et al., “Synthesis of Cluster Galactosides with High Affinity for the Hepatic Asialoglycoprotein Receptor” J. Med. Chem. (1995) 38:1538-1546, LEE et al., “New and more efficient multivalent glyco-ligands for asialoglycoprotein receptor of mammalian hepatocytes” Bioorganic & Medicinal Chemistry (2011) 19:2494-2500, RENSEN et al., “Determination of the Upper Size Limit for Uptake and Processing of Ligands by the Asialoglycoprotein Receptor on Hepatocytes in Vitro and in Vivo” J. Biol. Chem. (2001) 276(40):37577-37584, RENSEN et al., “Design and Synthesis of Novel N-Acetylgalactosamine-Terminated Glycolipids for Targeting of Lipoproteins to the Hepatic Asialoglycoprotein Receptor” J. Med. Chem. (2004) 47:5798-5808, SLIEDREGT et al., “Design and Synthesis of Novel Amphiphilic Dendritic Galactosides for Selective Targeting of Liposomes to the Hepatic Asialoglycoprotein Receptor” J. Med. Chem. (1999) 42:609-618, and Valentijn et al., “Solid-phase synthesis of lysine-based cluster galactosides with high affinity for the Asialoglycoprotein Receptor” Tetrahedron, 1997, 53(2), 759-770, each of which is incorporated by reference herein in its entirety.

In certain embodiments, conjugated antisense compounds comprise an RNase H based oligonucleotide (such as a gapmer) or a splice modulating oligonucleotide (such as a fully modified oligonucleotide) and any conjugate group comprising at least one, two, or three GalNAc groups. In certain embodiments a conjugated antisense compound comprises any conjugate group found in any of the following references: Lee, Carbohydr Res, 1978, 67, 509-514; Connolly et al., J Biol Chem, 1982, 257, 939-945; Pavia et al., Int J Pep Protein Res, 1983, 22, 539-548; Lee et al., Biochem, 1984, 23, 4255-4261; Lee et al., Glycoconjugate J, 1987, 4, 317-328; Toyokuni et al., Tetrahedron Lett, 1990, 31, 2673-2676; Biessen et al., J Med Chem, 1995, 38, 1538-1546; Valentijn et al., Tetrahedron, 1997, 53, 759-770; Kim et al., Tetrahedron Lett, 1997, 38, 3487-3490; Lee et al., Bioconjug Chem, 1997, 8, 762-765; Kato et al., Glycobiol, 2001, 11, 821-829; Rensen et al., J Biol Chem, 2001, 276, 37577-37584; Lee et al., Methods Enzymol, 2003, 362, 38-43; Westerlind et al., Glycoconj J, 2004, 21, 227-241; Lee et al., Bioorg Med Chem Lett, 2006, 16(19), 5132-5135; Maierhofer et al., Bioorg Med Chem, 2007, 15, 7661-7676; Khorev et al., Bioorg Med Chem, 2008, 16, 5216-5231; Lee et al., Bioorg Med Chem, 2011, 19, 2494-2500; Kornilova et al., Analyt Biochem, 2012, 425, 43-46; Pujol et al., Angew Chemie Int Ed Engl, 2012, 51, 7445-7448; Biessen et al., J Med Chem, 1995, 38, 1846-1852; Sliedregt et al., J Med Chem, 1999, 42, 609-618; Rensen et al., J Med Chem, 2004, 47, 5798-5808; Rensen et al., Arterioscler Thromb Vasc Biol, 2006, 26, 169-175; van Rossenberg et al., Gene Ther, 2004, 11, 457-464; Sato et al., J Am Chem Soc, 2004, 126, 14013-14022; Lee et al., J Org Chem, 2012, 77, 7564-7571; Biessen et al., FASEB J, 2000, 14, 1784-1792; Rajur et al., Bioconjug Chem, 1997, 8, 935-940; Duff et al., Methods Enzymol, 2000, 313, 297-321; Maier et al., Bioconjug Chem, 2003, 14, 18-29; Jayaprakash et al., Org Lett, 2010, 12, 5410-5413; Manoharan, Antisense Nucleic Acid Drug Dev, 2002, 12, 103-128; Merwin et al., Bioconjug Chem, 1994, 5, 612-620; Tomiya et al., Bioorg Med Chem, 2013, 21, 5275-5281; International applications WO1998/013381; WO2011/038356; WO1997/046098; WO2008/098788; WO2004/101619; WO2012/037254; WO2011/120053; WO2011/100131; WO2011/163121; WO2012/177947; WO2013/033230; WO2013/075035; WO2012/083185; WO2012/083046; WO2009/082607; WO2009/134487; WO2010/144740; WO2010/148013; WO1997/020563; WO2010/088537; WO2002/043771; WO2010/129709; WO2012/068187; WO2009/126933; WO2004/024757; WO2010/054406; WO2012/089352; WO2012/089602; WO2013/166121; WO2013/165816; U.S. Pat. Nos. 4,751,219; 8,552,163; 6,908,903; 7,262,177; 5,994,517; 6,300,319; 8,106,022; 7,491,805; 7,491,805; 7,582,744; 8,137,695; 6,383,812; 6,525,031; 6,660,720; 7,723,509; 8,541,548; 8,344,125; 8,313,772; 8,349,308; 8,450,467; 8,501,930; 8,158,601; 7,262,177; 6,906,182; 6,620,916; 8,435,491; 8,404,862; 7,851,615; Published U.S. Patent Application Publications US2011/0097264; US2011/0097265; US2013/0004427; US2005/0164235; US2006/0148740; US2008/0281044; US2010/0240730; US2003/0119724; US2006/0183886; US2008/0206869; US2011/0269814; US2009/0286973; US2011/0207799; US2012/0136042; US2012/0165393; US2008/0281041; US2009/0203135; US2012/0035115; US2012/0095075; US2012/0101148; US2012/0128760; US2012/0157509; US2012/0230938; US2013/0109817; US2013/0121954; US2013/0178512; US2013/0236968; US2011/0123520; US2003/0077829; US2008/0108801; and US2009/0203132; each of which is incorporated by reference in its entirety.

Cell Culture and Antisense Compounds Treatment

The effects of antisense compounds on the level, activity or expression of ANGPTL3 nucleic acids can be tested in vitro in a variety of cell types. Cell types used for such analyses are available from commercial vendors (e.g. American Type Culture Collection, Manassas, Va.; Zen-Bio, Inc., Research Triangle Park, N.C.; Clonetics Corporation, Walkersville, Md.) and cells are cultured according to the vendor's instructions using commercially available reagents (e.g. Invitrogen Life Technologies, Carlsbad, Calif.). Illustrative cell types include, but are not limited to, HepG2 cells, Hep3B cells, Huh7 (hepatocellular carcinoma) cells, primary hepatocytes, A549 cells, GM04281 fibroblasts and LLC-MK2 cells.

In Vitro Testing of Antisense Oligonucleotides

Described herein are methods for treatment of cells with antisense oligonucleotides, which can be modified appropriately for treatment with other antisense compounds.

In general, cells are treated with antisense oligonucleotides when the cells reach approximately 60-80% confluence in culture.

One reagent commonly used to introduce antisense oligonucleotides into cultured cells includes the cationic lipid transfection reagent LIPOFECTIN® (Invitrogen, Carlsbad, Calif.). Antisense oligonucleotides are mixed with LIPOFECTIN® in OPTI-MEM® 1 (Invitrogen, Carlsbad, Calif.) to achieve the desired final concentration of antisense oligonucleotide and a LIPOFECTIN® concentration that typically ranges 2 to 12 ug/mL per 100 nM antisense oligonucleotide.

Another reagent used to introduce antisense oligonucleotides into cultured cells includes LIPOFECTAMINE 2000® (Invitrogen, Carlsbad, Calif.). Antisense oligonucleotide is mixed with LIPOFECTAMINE 2000® in OPTI-MEM® 1 reduced serum medium (Invitrogen, Carlsbad, Calif.) to achieve the desired concentration of antisense oligonucleotide and a LIPOFECTAMINE® concentration that typically ranges 2 to 12 ug/mL per 100 nM antisense oligonucleotide.

Another reagent used to introduce antisense oligonucleotides into cultured cells includes Cytofectin® (Invitrogen, Carlsbad, Calif.). Antisense oligonucleotide is mixed with Cytofectin® in OPTI-MEM® 1 reduced serum medium (Invitrogen, Carlsbad, Calif.) to achieve the desired concentration of antisense oligonucleotide and a Cytofectin® concentration that typically ranges 2 to 12 ug/mL per 100 nM antisense oligonucleotide.

Another reagent used to introduce antisense oligonucleotides into cultured cells includes Oligofectamine™ (Invitrogen Life Technologies, Carlsbad, Calif.). Antisense oligonucleotide is mixed with Oligofectamine™ in Opti-MEM™-1 reduced serum medium (Invitrogen Life Technologies, Carlsbad, Calif.) to achieve the desired concentration of oligonucleotide with an Oligofectamine™ to oligonucleotide ratio of approximately 0.2 to 0.8 μL per 100 nM.

Another reagent used to introduce antisense oligonucleotides into cultured cells includes FuGENE 6 (Roche Diagnostics Corp., Indianapolis, Ind.). Antisense oligomeric compound was mixed with FuGENE 6 in 1 mL of serum-free RPMI to achieve the desired concentration of oligonucleotide with a FuGENE 6 to oligomeric compound ratio of 1 to 4 μL of FuGENE 6 per 100 nM.

Another technique used to introduce antisense oligonucleotides into cultured cells includes electroporation (Sambrook and Russell, Molecular Cloning: A Laboratory Manual, 3^(rd) Ed., 2001).

Cells are treated with antisense oligonucleotides by routine methods. Cells are typically harvested 16-24 hours after antisense oligonucleotide treatment, at which time RNA or protein levels of target nucleic acids are measured by methods known in the art and described herein. In general, when treatments are performed in multiple replicates, the data are presented as the average of the replicate treatments.

The concentration of antisense oligonucleotide used varies from cell line to cell line. Methods to determine the optimal antisense oligonucleotide concentration for a particular cell line are well known in the art. Antisense oligonucleotides are typically used at concentrations ranging from 1 nM to 300 nM when transfected with LIPOFECTAMINE2000®, Lipofectin or Cytofectin. Antisense oligonucleotides are used at higher concentrations ranging from 625 to 20,000 nM when transfected using electroporation.

RNA Isolation

RNA analysis can be performed on total cellular RNA or poly(A)+ mRNA. Methods of RNA isolation are well known in the art (Sambrook and Russell, Molecular Cloning: A Laboratory Manual, 3^(rd) Ed., 2001). RNA is prepared using methods well known in the art, for example, using the TRIZOL® Reagent (Invitrogen, Carlsbad, Calif.) according to the manufacturer's recommended protocols.

Analysis of Inhibition of Target Levels or Expression

Inhibition of levels or expression of an ANGPTL3 nucleic acid can be assayed in a variety of ways known in the art (Sambrook and Russell, Molecular Cloning: A Laboratory Manual, 3^(rd) Ed., 2001). For example, target nucleic acid levels can be quantitated by, e.g., Northern blot analysis, competitive polymerase chain reaction (PCR), or quantitative real-time PCR. RNA analysis can be performed on total cellular RNA or poly(A)+ mRNA. Methods of RNA isolation are well known in the art. Northern blot analysis is also routine in the art. Quantitative real-time PCR can be conveniently accomplished using the commercially available ABI PRISM® 7600, 7700, or 7900 Sequence Detection System, available from PE-Applied Biosystems, Foster City, Calif. and used according to manufacturer's instructions.

Quantitative Real-Time PCR Analysis of Target RNA Levels

Quantitation of target RNA levels can be accomplished by quantitative real-time PCR using the ABI PRISM® 7600, 7700, or 7900 Sequence Detection System (PE-Applied Biosystems, Foster City, Calif.) according to manufacturer's instructions. Methods of quantitative real-time PCR are well known in the art.

Prior to real-time PCR, the isolated RNA is subjected to a reverse transcriptase (RT) reaction, which produces complementary DNA (cDNA) that is then used as the substrate for the real-time PCR amplification. The RT and real-time PCR reactions are performed sequentially in the same sample well. RT and real-time PCR reagents are obtained from Invitrogen (Carlsbad, Calif.). RT and real-time-PCR reactions are carried out by methods well known to those skilled in the art.

Gene (or RNA) target quantities obtained by real time PCR can be normalized using either the expression level of a gene whose expression is constant, such as cyclophilin A or GADPH or by quantifying total RNA using RIBOGREEN® (Life Technologies™, Inc. Carlsbad, Calif.). Cyclophilin A or GADPH expression can be quantified by real time PCR, by being run simultaneously with the target, multiplexing, or separately. Total RNA can be quantified using RIBOGREEN® RNA quantification reagent. Methods of RNA quantification by RIBOGREEN® are taught in Jones, L. J., et al, (Analytical Biochemistry, 1998, 265, 368-374). A CYTOFLUOR® 4000 instrument (PE Applied Biosystems) can be used to measure RIBOGREEN® fluorescence.

Methods for designing real-time PCR probes and primers are well known in the art, and can include the use of software such as PRIMER EXPRESS® Software (Applied Biosystems, Foster City, Calif.). Probes and primers used in real-time PCR were designed to hybridize to ANGPTL3 specific sequences and are disclosed in the Examples below. The target specific PCR probes can have FAM covalently linked to the 5′ end and TAMRA or MGB covalently linked to the 3′ end, where FAM is the fluorescent dye and TAMRA or MGB is the quencher dye.

Analysis of Protein Levels

Antisense inhibition of ANGPTL3 nucleic acids can be assessed by measuring ANGPTL3 protein levels. Protein levels of ANGPTL3 can be evaluated or quantitated in a variety of ways well known in the art, such as immunoprecipitation, Western blot analysis (immunoblotting), enzyme-linked immunosorbent assay (ELISA), quantitative protein assays, protein activity assays (for example, caspase activity assays), immunohistochemistry, immunocytochemistry or fluorescence-activated cell sorting (FACS) (Sambrook and Russell, Molecular Cloning: A Laboratory Manual, 3^(rd) Ed., 2001). Antibodies directed to a target can be identified and obtained from a variety of sources, such as the MSRS catalog of antibodies (Aerie Corporation, Birmingham, Mich.), or can be prepared via conventional monoclonal or polyclonal antibody generation methods well known in the art.

In Vivo Testing of Antisense Compounds

Antisense compounds, for example, antisense oligonucleotides, are tested in animals to assess their ability to inhibit expression of ANGPTL3 and produce phenotypic changes. Testing can be performed in normal animals, or in experimental disease models. For administration to animals, antisense oligonucleotides are formulated in a pharmaceutically acceptable diluent, such as phosphate-buffered saline. Administration includes parenteral routes of administration. Following a period of treatment with antisense oligonucleotides, RNA is isolated from tissue and changes in ANGPTL3 nucleic acid expression are measured. Changes in ANGPTL3 protein levels are also measured.

Certain Indications

In certain embodiments, provided herein are methods of treating an individual comprising administering one or more pharmaceutical compositions as described herein. In certain embodiments, the individual has a metabolic disease and/or cardiovascular disease. In certain embodiments, the individual has combined hyperlipidemia (e.g., familial or non-familial), hypercholesterolemia (e.g., familial homozygous hypercholesterolemia (HoFH), familial heterozygous hypercholesterolemia (HeFH)), dyslipidemia, lipodystrophy, hypertriglyceridemia (e.g., heterozygous LPL deficiency, homozygous LPL deficiency), coronary artery disease (CAD), familial chylomicronemia syndrome (FCS), hyperlipoproteinemia Type IV), metabolic syndrome, non-alcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH), diabetes (e.g., Type 2 diabetes, Type 2 diabetes with dyslipidemia), insulin resistance (e.g., insulin resistance with dyslipidemia), vascular wall thickening, high blood pressure (e.g., pulmonary arterial hypertension), sclerosis (e.g., atherosclerosis, systemic sclerosis, progressive skin sclerosis and proliferative obliterative vasculopathy such as digital ulcers and pulmonary vascular involvement), or a combination thereof.

In certain embodiments, the compounds targeted to ANGPTL3 described herein modulate lipid and/or energy metabolism in an animal. In certain embodiments, the compounds targeted to ANGPTL3 described herein modulate physiological markers or phenotypes of hypercholesterolemia, dyslipidemia, lipodystrophy, hypertriglyceridemia, metabolic syndrome, NAFLD, NASH and/or diabetes. For example, administration of the compounds to animals can modulate one or more of VLDL, non-esterified fatty acids (NEFA), LDL, cholesterol, triglyceride, glucose, insulin or ANGPTL3 levels. In certain embodiments, the modulation of the physiological markers or phenotypes can be associated with inhibition of ANGPTL3 by the compounds.

In certain embodiments, the compounds targeted to ANGPTL3 described herein reduce and/or prevent one or more of hepatic TG accumulation (i.e. hepatic steatosis), atherosclerosis, vascular wall thinkening (e.g., arterial intima-media thickening), combined hyperlipidemia (e.g., familial or non-familial), hypercholesterolemia (e.g., familial homozygous hypercholesterolemia (HoFH), familial heterozygous hypercholesterolemia (HeFH)), dyslipidemia, lipodystrophy, hypertriglyceridemia (e.g., heterozygous LPL deficiency, homozygous LPL deficiency, familial chylomicronemia syndrome (FCS), hyperlipoproteinemia Type IV), metabolic syndrome, non-alcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH), diabetes (e.g., Type 2 diabetes, Type 2 diabetes with dyslipidemia), insulin resistance (e.g., insulin resistance with dyslipidemia), high blood pressure and sclerosis, or any combination thereof. In certain embodiments, the compounds targeted to ANGPTL3 described herein improve insulin sensitivity.

In certain embodiments, administration of an antisense compound targeted to an ANGPTL3 nucleic acid results in reduction of ANGPTL3 expression by about at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99%, or a range defined by any two of these values.

In certain embodiments, pharmaceutical compositions comprising an antisense compound targeted to ANGPTL3 are used for the preparation of a medicament for treating a patient suffering from, or susceptible to, a metabolic disease or cardiovascular disease. In certain embodiments, pharmaceutical compositions comprising an antisense compound targeted to ANGPTL3 are used in the preparation of a medicament for treating a patient suffering from, or susceptible to, one or more of combined hyperlipidemia (e.g., familial or non-familial), hypercholesterolemia (e.g., familial homozygous hypercholesterolemia (HoFH), familial heterozygous hypercholesterolemia (HeFH)), dyslipidemia, lipodystrophy, hypertriglyceridemia (e.g., familial chylomicronemia syndrome (FCS), hyperlipoproteinemia Type IV), metabolic syndrome, non-alcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH), diabetes (e.g., Type 2 diabetes, Type 2 diabetes with dyslipidemia), insulin resistance (e.g., insulin resistance with dyslipidemia), vascular wall thickening, high blood pressure and sclerosis, or a combination thereof.

Administration

In certain embodiments, the compounds and compositions as described herein are administered parenterally.

In certain embodiments, parenteral administration is by infusion. Infusion can be chronic or continuous or short or intermittent. In certain embodiments, infused pharmaceutical agents are delivered with a pump.

In certain embodiments, parenteral administration is by injection. The injection can be delivered with a syringe or a pump. In certain embodiments, the injection is a bolus injection. In certain embodiments, the injection is administered directly to a tissue or organ. In certain embodiments, the injection is subcutaneous.

Certain Combination Therapies

In certain embodiments, a first agent comprising the modified oligonucleotide disclosed herein is co-administered with one or more secondary agents. In certain embodiments, such second agents are designed to treat the same disease, disorder or condition as the first agent described herein. In certain embodiments, such second agents are designed to treat a different disease, disorder, or condition as the first agent described herein. In certain embodiments, such second agents are designed to treat an undesired side effect of one or more pharmaceutical compositions as described herein. In certain embodiments, second agents are co-administered with the first agent to treat an undesired effect of the first agent. In certain embodiments, second agents are co-administered with the first agent to produce a combinational effect. In certain embodiments, second agents are co-administered with the first agent to produce a synergistic effect.

In certain embodiments, a first agent and one or more second agents are administered at the same time. In certain embodiments, the first agent and one or more second agents are administered at different times. In certain embodiments, the first agent and one or more second agents are prepared together in a single pharmaceutical formulation. In certain embodiments, the first agent and one or more second agents are prepared separately.

In certain embodiments, second agents include, but are not limited to a glucose-lowering agent or a lipid-lowering agent. The glucose lowering agent can include, but is not limited to, a therapeutic lifestyle change, PPAR agonist, a dipeptidyl peptidase (IV) inhibitor, a GLP-1 analog, insulin or an insulin analog, an insulin secretagogue, a SGLT2 inhibitor, a human amylin analog, a biguanide, an alpha-glucosidase inhibitor, or a combination thereof. The glucose-lowering agent can include, but is not limited to metformin, sulfonylurea, rosiglitazone, meglitinide, thiazolidinedione, alpha-glucosidase inhibitor or a combination thereof. The sulfonylurea can be acetohexamide, chlorpropamide, tolbutamide, tolazamide, glimepiride, a glipizide, a glyburide, or a gliclazide. The meglitinide can be nateglinide or repaglinide. The thiazolidinedione can be pioglitazone or rosiglitazone. The alpha-glucosidase can be acarbose or miglitol. In certain embodiments the lipid lowering therapy can include, but is not limited to, a therapeutic lifestyle change, niacin, HMG-CoA reductase inhibitor, cholesterol absorption inhibitor, MTP inhibitor (e.g., a small molecule, polypeptide, antibody or antisense compound targeted to MTP), fibrate, PCSK9 inhibitor (e.g., PCSK9 antibodies, polypeptides, small molecules nucleic acid compounds targeting PCSK9), CETP inhibitor (e.g., small molecules such as torcetrapib and anacetrapib, polypeptides, antibodies or nucleic acid compounds targeted to CETP), apoC-III inhibitor (e.g., a small molecule, polypeptide, antibody or nucleic acid compounds targeted to apoC-III), apoB inhibitor (e.g., a small molecule, polypeptide, antibody or nucleic acid compounds targeted to apoB), beneficial oils rich in omega-3 fatty acids, omega-3 fatty acids or any combination thereof. The HMG-CoA reductase inhibitor can be atorvastatin, rosuvastatin, fluvastatin, lovastatin, pravastatin, simvastatin and the like. The cholesterol absorption inhibitor can be ezetimibe. The fibrate can be fenofibrate, bezafibrate, ciprofibrate, clofibrate, gemfibrozil and the like. The beneficial oil rich in omega-3 fatty acids can be krill, fish (e.g., Vascepa®), flaxseed oil and the like. The omega-3 fatty acid can be ALA, DHA, EPA and the like.

Certain Compounds

Antisense oligonucleotides targeting human ANGPTL3 were described in an earlier publication (see PCT Patent Publication No. WO 2011/085271 published Jul. 14, 2011, incorporated by reference herein, in its entirety). Several oligonucleotides (233676, 233690, 233710, 233717, 233721, 233722, 337459, 337460, 337474, 337477, 337478, 337479, 337481, 337484, 337487, 337488, 337490, 337491, 337492, 337497, 337498, 337503, 337505, 337506, 337508, 337513, 337514, 337516, 337520, 337521, 337525, 337526 and 337528) described therein, including the top ten most potent antisense compounds in vitro, were used as benchmarks throughout select in vitro screens for antisense compounds described hereinbelow and in U.S. Ser. No. 61/920,652. Of the most potent compounds described in WO 2011/085271, ISIS 233722 was found to be highly variable in its ability to inhibit ANGPTL3. According, although initially included in some in vitro studies, 233722 was not selected as a benchmark for further studies. Of the previously identified potent in vitro benchmark compounds, five (233710, 233717, 337477, 337478, 337479 and 337487) were selected for testing in vivo, as described hereinbelow, in huANGPTL3 transgenic mice to assess the most potent in reducing human mRNA transcript and protein expression (Example 126). The antisense oligonucleotide with the highest initial in vivo potency in reducing ANGPTL3 levels (233710) was used as a benchmark for in vivo assessment of the new antisense compounds described hereinbelow.

In certain embodiments, the antisense compounds described herein benefit from one or more improved properties relative to the antisense compounds described in WO 2011/085271 and in U.S. Ser. No. 61/920,652. These improved properties are demonstrated in the examples herein, and a non-exhaustive summary of the examples is provided below for ease of reference.

In a first screen described herein, about 3000 newly designed 5-10-5 MOE gapmer antisense compounds targeting human ANGPTL3 were tested in Hep3B cells for their effect on human ANGPTL3 mRNA in vitro (Example 116). The mRNA inhibition levels of the new antisense compounds were assessed with some previously designed antisense compounds (233717, 337484, 337487, 337492 and 337516) used as benchmarks in select studies. Of the about 3000 newly designed antisense compounds from this first screen, about 85 antisense compounds were selected for in vitro dose-dependent inhibition studies to determine their half maximal inhibitory concentration (IC₅₀) (Examples 117-118). Of the about 85 new antisense compounds tested for their half maximal inhibitory concentration (IC₅₀), about 38 antisense compounds that demonstrated potent dose-dependent reduction of ANGPTL3 were selected for in vivo potency and tolerability (ALT and AST) testing in mice (Examples 126-127) with antisense compound 233710 used as a benchmark.

In a second screen described herein, about 2000 newly designed antisense compounds targeting human ANGPTL3 with a MOE gapmer motif or a mixed motif (deoxy, 5-10-5 MOE and cET gapmers) were also tested in Hep3B cells for their effect on human ANGPTL3 mRNA in vitro (Examples 119-121). The inhibition levels of the new antisense compounds were assessed with some previously designed antisense compounds (233717, 337487, 337513, 337514 and 337516) used as benchmarks in select studies. Of the about 2000 newly designed antisense compounds from this second screen, about 147 antisense compounds were selected for in vitro dose-dependent inhibition studies to determine their half maximal inhibitory concentration (IC₅₀) (Examples 122-125). Of the about 147 new antisense compounds from tested for their half maximal inhibitory concentration (IC₅₀), about 73 antisense compounds that demonstrated potent dose-dependent reduction of ANGPTL3 were selected for in vivo potency and tolerability (e.g., ALT and AST) testing in mice (Examples 126-127) with antisense compound 233710 used as a benchmark.

Of the about 111 antisense compounds from screens one and two that were tested for potency and tolerability in mice, 24 were selected for more extensive tolerability testing in mice by assessing liver metabolic markers, such as alanine transaminase (ALT), aspartate transaminase (AST), albumin and bilirubin, as well as kidney metabolic markers BUN and creatinine and organ weight (Example 127).

In parallel with the in vivo murine studies seventeen antisense compounds were selected for viscosity testing (Example 128). Generally, antisense compounds that were not optimal for viscosity were not taken forward in further studies.

Based on the results of the mice tolerability study, twenty antisense compounds were selected for in vivo tolerability testing in rats (Example 129). In the rats, liver metabolic markers, such as ALT, AST, albumin and bilirubin, body and organ weights, as well as kidney metabolic markers, such as BUN, creatinine and total protein/creatinine ratio, were measured to determine the tolerability of a compound in vivo.

The twenty antisense compounds tested in the rats were also assessed for cross-reactivity to a rhesus monkey ANGPTL3 gene sequence (Example 130). Although the antisense compounds in this study were tested in cynomolgus monkeys, the cynomolgus monkey ANGPTL3 sequence was not available for comparison to the sequences of the full-length compounds, therefore the sequences of the antisense compounds were compared to that of the closely related rhesus monkey. The sequences of eight antisense compounds were found to have 0-2 mismatches with the rhesus ANGPTL3 gene sequence and were further studied in cynomolgus monkeys (Example 130). The eight antisense compounds (ISIS 563580, ISIS 560400, ISIS 567320, ISIS 567321, ISIS 544199, ISIS 567233, ISIS 561011 and ISIS 559277) were tested for inhibition of ANGPTL3 mRNA and protein expression as well as tolerability in the monkeys. In the tolerability studies, body weights, liver metabolic markers (ALT, AST and bilirubin), kidney metabolic markers (BUN and creatinine), hematology parameters (blood cell counts, hemoglobin and hematocrit), and pro-inflammatory markers (CRP and C3) were measured. Additionally, the full-length oligonucleotide concentration present in liver and kidney was measured and the ratio of full-length oligonucleotide in the kidney/liver was calculated.

The sequence of a potent and tolerable antisense compound, ISIS 563580, assessed in cynomolgus monkeys was further modified with a GalNAc conjugate and/or changes in the backbone chemistry as shown in Examples 113-115 and 131 and evaluated for increase potency.

Accordingly, provided herein are antisense compounds with any one or more improved characteristics e.g., improved relative to the antisense compounds described in WO 2011/085271 and in U.S. Ser. No. 61/920,652. In certain embodiments, provided herein are antisense compounds comprising a modified oligonucleotide as described herein targeted to, or specifically hybridizable with, a region of nucleotides of any one of SEQ ID NOs: 1-2.

In certain embodiments, certain antisense compounds as described herein are efficacious by virtue of their potency in inhibiting ANGPTL3 expression. In certain embodiments, the compounds or compositions inhibit ANGPTL3 by at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95%.

In certain embodiments, certain antisense compounds as described herein are efficacious by virtue of an in vitro IC₅₀ of less than 20 μM, less than 10 μM, less than 8 μM, less than 5 μM, less than 2 μM, less than 1 μM, less than 0.9 μM, less than 0.8 μM, less than 0.7 μM, less than 0.6 μM, or less than 0.5 μM when tested in human cells, for example, in the Hep3B cell line (as described in Examples 117-118 and 122-125). In certain embodiments, preferred antisense compounds having an IC₅₀<1.0 μM include SEQ ID NOs: 15, 20, 24, 34, 35, 36, 37, 42, 43, 44, 47, 50, 51, 57, 58, 60, 77, 79, 82, 87, 88, 90, 91, 93, 94, 100, 101, 104, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 169, 170, 177, 188, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, and 232. In certain embodiments, preferred antisense compounds having an IC₅₀<0.9 μM include SEQ ID NOs: 15, 20, 35, 36, 42, 43, 44, 50, 57, 60, 77, 79, 87, 88, 90, 91, 93, 94, 101, 104, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 177, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, and 232. In certain embodiments, preferred antisense compounds having an IC₅₀<0.8 μM include SEQ ID NOs: 15, 20, 35, 36, 42, 43, 44, 50, 57, 60, 77, 79, 87, 88, 90, 91, 93, 94, 101, 104, 110, 111, 112, 113, 114, 115, 116, 117, 118, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 177, 209, 210, 211, 212, 213, 214, 215, 217, 218, 219, 220, 221, 222, 223, 224, 225, 228, 229, 230, 231, and 232. In certain embodiments, preferred antisense compounds having an IC₅₀<0.7 μM include SEQ ID NOs: 15, 20, 36, 42, 43, 57, 60, 114, 117, 127, 131, 177, 209, 210, 211, 212, 213, 214, 215, 217, 218, 219, 220, 221, 222, 223, 224, 225, 228, 229, 230, 231, and 232. In certain embodiments, preferred antisense compounds having an IC₅₀<0.6 μM include SEQ ID NOs: 15, 20, 36, 42, 43, 57, 60, 114, 117, 127, 131, 177, 209, 210, 211, 212, 213, 215, 217, 218, 219, 220, 221, 222, 224, 225, 228, 229, 230, 231, and 232. In certain embodiments, preferred antisense compounds having an IC₅₀<0.5 μM include SEQ ID NOs: 43, 114, 117, 127, 131, 177, 209, 210, 211, 212, 215, 217, 218, 219, 220, 221, 222, 229, 230, and 232.

In certain embodiments, certain antisense compounds as described herein are efficacious by virtue of having a viscosity of less than 40 cP, less than 35 cP, less than 30 cP, less than 25 cP, less than 20 cP, less than 15 cP, or less than 10 cP when measured by an assay (as described in Example 128). Oligonucleotides having a viscosity greater than 40 cP would have less than optimal viscosity. In certain embodiments, preferred antisense compounds having a viscosity <20 cP include SEQ ID NOs: 16, 18, 20, 34, 35, 36, 38, 49, 77, 90, 93, and 94. In certain embodiments, preferred antisense compounds having a viscosity <15 cP include SEQ ID NOs: 16, 18, 20, 34, 35, 38, 49, 90, 93, and 94. In certain embodiments, preferred antisense compounds having a viscosity <10 cP include SEQ ID NOs: 18, 34, 35, 49, 90, 93, and 94.

In certain embodiments, certain antisense compounds as described herein are highly tolerable, as demonstrated by the in vivo tolerability measurements described in the examples. In certain embodiments, the certain antisense compounds as described herein are highly tolerable, as demonstrated by having an increase in ALT and/or AST value of no more than 3 fold, 2 fold or 1.5 fold over saline treated animals.

In certain embodiments, certain antisense compounds as described herein are efficacious by virtue of having one or more of an inhibition potency of greater than 50%, an in vitro IC₅₀ of less than 1 μM, a viscosity of less than 20 cP, and no more than a 3 fold increase in ALT and/or AST.

In certain embodiments, ISIS 563580 (SEQ ID NO: 77) is preferred. This compound was found to be a potent inhibitor in ANGPTL3 transgenic mice and the most tolerable antisense compound. It had an acceptable viscosity of about 16.83 cP and an IC₅₀ value of <0.8 μM in vitro. In mice it had no more than a 3 fold increase in ALT and/or AST levels over saline treated animals. Also, in monkeys, it was among the most tolerable and potent compounds in inhibiting ANGPTL3 and had the best ratio of full-length oligonucleotide concentration.

In certain embodiments, ISIS 544199 (SEQ ID NO: 20) is preferred. This compound was found to be a potent and tolerable antisense compound. It had an acceptable viscosity of 1.7 cP and an IC₅₀ value of <0.5 μM in vitro. In mice it had no more than a 3 fold increase in ALT and/or AST levels over saline treated animals. Also, in monkeys, it was among the most potent compounds in inhibiting ANGPTL3 and had a good ratio of full-length oligonucleotide concentration.

In certain embodiments, ISIS 559277 (SEQ ID NO: 110) is preferred. This compound was found to be a potent and tolerable antisense compound. It had an IC₅₀ value of <0.8 μM in vitro. In mice it had no more than a 3 fold increase in ALT and/or AST levels over saline treated animals. Also, in monkeys, it was among the most potent compounds in inhibiting ANGPTL3 and had a good ratio of full-length oligonucleotide concentration.

In certain embodiments, a GalNAc conjugated antisense compound, ISIS 658501 (SEQ ID NO: 4912), is preferred. This antisense compound was found to be more potent than its parent compound ISIS 563580 (SEQ ID NO: 77) as shown by the inhibition levels.

In certain embodiments, a GalNAc conjugated antisense compound, ISIS 703801 (SEQ ID NO: 77), is preferred. This antisense compound was found to be several fold more potent than its parent compound ISIS 563580 (SEQ ID NO: 77). ISIS 703801 had an ID50 value of 1 while ISIS 563580 had an ID50 value of 6.

In certain embodiments, a GalNAc conjugated antisense compound, ISIS 703802 (SEQ ID NO: 77), is preferred. This antisense compound was found to be several fold more potent than its parent compound ISIS 563580 (SEQ ID NO: 77). ISIS 703802 had an ID50 value of 0.3 while ISIS 563580 had an ID50 value of 6.

EXAMPLES

The following examples illustrate certain embodiments of the present disclosure and are not limiting. Moreover, where specific embodiments are provided, the inventors have contemplated generic application of those specific embodiments. For example, disclosure of an oligonucleotide having a particular motif provides reasonable support for additional oligonucleotides having the same or similar motif. And, for example, where a particular high-affinity modification appears at a particular position, other high-affinity modifications at the same position are considered suitable, unless otherwise indicated.

Example 1: General Method for the Preparation of Phosphoramidites, Compounds 1, 1a and 2

-   -   Bx is a heterocyclic base;

Compounds 1, 1a and 2 were prepared as per the procedures well known in the art as described in the specification herein (see Seth et al., Bioorg. Med. Chem., 2011, 21(4), 1122-1125, J. Org. Chem., 2010, 75(5), 1569-1581, Nucleic Acids Symposium Series, 2008, 52(1), 553-554); and also see published PCT International Applications (WO 2011/115818, WO 2010/077578, WO2010/036698, WO2009/143369, WO 2009/006478, and WO 2007/090071), and U.S. Pat. No. 7,569,686).

Example 2: Preparation of Compound 7

Compounds 3 (2-acetamido-1,3,4,6-tetra-O-acetyl-2-deoxy-β-Dgalactopyranose or galactosamine pentaacetate) is commercially available. Compound 5 was prepared according to published procedures (Weber et al., J. Med. Chem., 1991, 34, 2692).

Example 3: Preparation of Compound 11

Compounds 8 and 9 are commercially available.

Example 4: Preparation of Compound 18

Compound 11 was prepared as per the procedures illustrated in Example 3. Compound 14 is commercially available. Compound 17 was prepared using similar procedures reported by Rensen et al., J. Med. Chem., 2004, 47, 5798-5808.

Example 5: Preparation of Compound 23

Compounds 19 and 21 are commercially available.

Example 6: Preparation of Compound 24

Compounds 18 and 23 were prepared as per the procedures illustrated in Examples 4 and 5.

Example 7: Preparation of Compound 25

Compound 24 was prepared as per the procedures illustrated in Example 6.

Example 8: Preparation of Compound 26

Compound 24 is prepared as per the procedures illustrated in Example 6.

Example 9: General Preparation of Conjugated ASOs Comprising GalNAc₃-1 at the 3′ Terminus, Compound 29

-   -   Wherein the protected GalNAc₃-1 has the structure:

The GalNAc₃ cluster portion of the conjugate group GalNAc₃-1 (GalNAc₃-1_(a)) can be combined with any cleavable moiety to provide a variety of conjugate groups. Wherein GalNAc₃-1_(a) has the formula:

The solid support bound protected GalNAc₃-1, Compound 25, was prepared as per the procedures illustrated in Example 7. Oligomeric Compound 29 comprising GalNAc₃-1 at the 3′ terminus was prepared using standard procedures in automated DNA/RNA synthesis (see Dupouy et al., Angew. Chem. Int. Ed., 2006, 45, 3623-3627). Phosphoramidite building blocks, Compounds 1 and 1a were prepared as per the procedures illustrated in Example 1. The phosphoramidites illustrated are meant to be representative and not intended to be limiting as other phosphoramidite building blocks can be used to prepare oligomeric compounds having a predetermined sequence and composition. The order and quantity of phosphoramidites added to the solid support can be adjusted to prepare gapped oligomeric compounds as described herein. Such gapped oligomeric compounds can have predetermined composition and base sequence as dictated by any given target.

Example 10: General Preparation Conjugated ASOs Comprising GalNAc₃-1 at the 5′ Terminus, Compound 34

The Unylinker™ 30 is commercially available. Oligomeric Compound 34 comprising a GalNAc₃-1 cluster at the 5′ terminus is prepared using standard procedures in automated DNA/RNA synthesis (see Dupouy et al., Angew. Chem. Int. Ed., 2006, 45, 3623-3627). Phosphoramidite building blocks, Compounds 1 and 1a were prepared as per the procedures illustrated in Example 1. The phosphoramidites illustrated are meant to be representative and not intended to be limiting as other phosphoramidite building blocks can be used to prepare an oligomeric compound having a predetermined sequence and composition. The order and quantity of phosphoramidites added to the solid support can be adjusted to prepare gapped oligomeric compounds as described herein. Such gapped oligomeric compounds can have predetermined composition and base sequence as dictated by any given target.

Example 11: Preparation of Compound 39

Compounds 4, 13 and 23 were prepared as per the procedures illustrated in Examples 2, 4, and 5. Compound 35 is prepared using similar procedures published in Rouchaud et al., Eur. J. Org. Chem., 2011, 12, 2346-2353.

Example 12: Preparation of Compound 40

Compound 38 is prepared as per the procedures illustrated in Example 11.

Example 13: Preparation of Compound 44

Compounds 23 and 36 are prepared as per the procedures illustrated in Examples 5 and 11. Compound 41 is prepared using similar procedures published in WO 2009082607.

Example 14: Preparation of Compound 45

Compound 43 is prepared as per the procedures illustrated in Example 13.

Example 15: Preparation of Compound 47

Compound 46 is commercially available.

Example 16: Preparation of Compound 53

Compounds 48 and 49 are commercially available. Compounds 17 and 47 are prepared as per the procedures illustrated in Examples 4 and 15.

Example 17: Preparation of Compound 54

Compound 53 is prepared as per the procedures illustrated in Example 16.

Example 18: Preparation of Compound 55

Compound 53 is prepared as per the procedures illustrated in Example 16.

Example 19: General Method for the Preparation of Conjugated ASOs Comprising GalNAc₃-1 at the 3′ Position Via Solid Phase Techniques (Preparation of ISIS 647535, 647536 and 651900)

Unless otherwise stated, all reagents and solutions used for the synthesis of oligomeric compounds are purchased from commercial sources. Standard phosphoramidite building blocks and solid support are used for incorporation nucleoside residues which include for example T, A, G, and C residues. A 0.1 μM solution of phosphoramidite in anhydrous acetonitrile was used for β-D-2′-deoxyribonucleoside and 2′-MOE.

The ASO syntheses were performed on ABI 394 synthesizer (1-2 μmol scale) or on GE Healthcare Bioscience ÄKTA oligopilot synthesizer (40-200 μmol scale) by the phosphoramidite coupling method on an GalNAc₃-1 loaded VIMAD solid support (110 μmol/g, Guzaev et al., 2003) packed in the column. For the coupling step, the phosphoramidites were delivered 4 fold excess over the loading on the solid support and phosphoramidite condensation was carried out for 10 min. All other steps followed standard protocols supplied by the manufacturer. A solution of 6% dichloroacetic acid in toluene was used for removing dimethoxytrityl (DMT) group from 5′-hydroxyl group of the nucleotide. 4,5-Dicyanoimidazole (0.7 M) in anhydrous CH₃CN was used as activator during coupling step. Phosphorothioate linkages were introduced by sulfurization with 0.1 μM solution of xanthane hydride in 1:1 pyridine/CH₃CN for a contact time of 3 minutes. A solution of 20% tert-butylhydroperoxide in CH₃CN containing 6% water was used as an oxidizing agent to provide phosphodiester internucleoside linkages with a contact time of 12 minutes.

After the desired sequence was assembled, the cyanoethyl phosphate protecting groups were deprotected using a 1:1 (v/v) mixture of triethylamine and acetonitrile with a contact time of 45 minutes. The solid-support bound ASOs were suspended in aqueous ammonia (28-30 wt %) and heated at 55° C. for 6 h.

The unbound ASOs were then filtered and the ammonia was boiled off. The residue was purified by high pressure liquid chromatography on a strong anion exchange column (GE Healthcare Bioscience, Source 30Q, 30 μm, 2.54×8 cm, A=100 mM ammonium acetate in 30% aqueous CH₃CN, B=1.5 μM NaBr in A, 0-40% of B in 60 min, flow 14 mL min-1, X=260 nm). The residue was desalted by HPLC on a reverse phase column to yield the desired ASOs in an isolated yield of 15-30% based on the initial loading on the solid support. The ASOs were characterized by ion-pair-HPLC coupled MS analysis with Agilent 1100 MSD system.

Antisense oligonucleotides not comprising a conjugate were synthesized using standard oligonucleotide synthesis procedures well known in the art.

Using these methods, three separate antisense compounds targeting ApoC III were prepared. As summarized in Table 17, below, each of the three antisense compounds targeting ApoC III had the same nucleobase sequence; ISIS 304801 is a 5-10-5 MOE gapmer having all phosphorothioate linkages; ISIS 647535 is the same as ISIS 304801, except that it had a GalNAc₃-1 conjugated at its 3′ end; and ISIS 647536 is the same as ISIS 647535 except that certain internucleoside linkages of that compound are phosphodiester linkages. As further summarized in Table 17, two separate antisense compounds targeting SRB-1 were synthesized. ISIS 440762 was a 2-10-2 cEt gapmer with all phosphorothioate internucleoside linkages; ISIS 651900 is the same as ISIS 440762, except that it included a GalNAc₃-1 at its 3′-end.

TABLE 17 Modified ASO targeting ApoC III and SRB-1 SEQ CalCd Observed ID ASO Sequence (5′ to 3′) Target Mass Mass No. ISIS A_(es)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(ds)T_(ds)T_(ds)G_(ds)T_(ds) ^(m)C_(ds) ^(m)C_(ds)A_(ds)G_(ds) ^(m)C_(ds)T_(es)T_(es)T_(es)A_(es)T_(e) ApoC 7165.4 7164.4 4878 304801 III ISIS A_(es)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(ds)T_(ds)T_(ds)G_(ds)T_(ds) ^(m)C_(ds) ^(m)C_(ds)A_(ds)G_(ds) ^(m)C_(ds)T_(es)T_(es)T_(es)A_(es)T_(eo) A _(do′) - ApoC 9239.5 9237.8 4879 647535 GalNAc ₃ -1 _(a) III ISIS A_(es)G_(eo) ^(m)C_(eo)T_(eo)T_(eo) ^(m)C_(ds)T_(ds)T_(ds)G_(ds)T_(ds) ^(m)C_(ds) ^(m)C_(ds)A_(ds)G_(ds) ^(m)C_(ds)T_(eo)T_(eo)T_(es)A_(es)T_(eo) A _(do′) - ApoC 9142.9 9140.8 4879 647536 GalNAc ₃ -1 _(a) III ISIS T_(ks) ^(m)C_(ks)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(ks) ^(m)C_(k) SRB-1 4647.0 4646.4 4880 440762 ISIS T_(ks) ^(m)C_(ks)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(ks) ^(m)C_(ko) A _(do′) -GalNAc ₃ -1 _(a) SRB-1 6721.1 6719.4 4881 651900 Subscripts: “e” indicates 2′-MOE modified nucleoside; “d” indicates β-D-2′-deoxyribonucleoside; “k” indicates 6′-(S)—CH₃ bicyclic nucleoside (e.g. cEt); “s” indicates phosphorothioate internucleoside linkages (PS); “o” indicates phosphodiester internucleoside linkages (PO); and “o′” indicates —O—P(═O)(OH)—. Superscript “m” indicates 5-methylcytosines. “GalNAc₃-1” indicates a conjugate group having the structure shown previously in Example 9. Note that GalNAc₃-1 comprises a cleavable adenosine which links the ASO to remainder of the conjugate, which is designated “GalNAc₃-1_(a).” This nomenclature is used in the above table to show the full nucleobase sequence, including the adenosine, which is part of the conjugate. Thus, in the above table, the sequences could also be listed as ending with “GalNAc₃-1” with the “Ado” omitted. This convention of using the subscript “a” to indicate the portion of a conjugate group lacking a cleavable nucleoside or cleavable moiety is used throughout these Examples. This portion of a conjugate group lacking the cleavable moiety is referred to herein as a “cluster” or “conjugate cluster” or “GalNAc₃ cluster.” In certain instances it is convenient to describe a conjugate group by separately providing its cluster and its cleavable moiety.

Example 20: Dose-Dependent Antisense Inhibition of Human ApoC III in huApoC III Transgenic Mice

ISIS 304801 and ISIS 647535, each targeting human ApoC III and described above, were separately tested and evaluated in a dose-dependent study for their ability to inhibit human ApoC III in human ApoC III transgenic mice.

Treatment

Human ApoCIII transgenic mice were maintained on a 12-hour light/dark cycle and fed ad libitum Teklad lab chow. Animals were acclimated for at least 7 days in the research facility before initiation of the experiment. ASOs were prepared in PBS and sterilized by filtering through a 0.2 micron filter. ASOs were dissolved in 0.9% PBS for injection.

Human ApoC III transgenic mice were injected intraperitoneally once a week for two weeks with ISIS 304801 or 647535 at 0.08, 0.25, 0.75, 2.25 or 6.75 μmol/kg or with PBS as a control. Each treatment group consisted of 4 animals. Forty-eight hours after the administration of the last dose, blood was drawn from each mouse and the mice were sacrificed and tissues were collected.

ApoC III mRNA Analysis

ApoC III mRNA levels in the mice's livers were determined using real-time PCR and RIBOGREEN® RNA quantification reagent (Molecular Probes, Inc. Eugene, Oreg.) according to standard protocols. ApoC III mRNA levels were determined relative to total RNA (using Ribogreen), prior to normalization to PBS-treated control. The results below are presented as the average percent of ApoC III mRNA levels for each treatment group, normalized to PBS-treated control and are denoted as “% PBS”. The half maximal effective dosage (ED₅₀) of each ASO is also presented in Table 18, below.

As illustrated, both antisense compounds reduced ApoC III RNA relative to the PBS control. Further, the antisense compound conjugated to GalNAc₃-1 (ISIS 647535) was substantially more potent than the antisense compound lacking the GalNAc₃-1 conjugate (ISIS 304801).

TABLE 18 Effect of ASO treatment on ApoC III mRNA levels in human ApoC III transgenic mice Dose % ED₅₀ 3′ Internucleoside SEQ ASO (μmol/kg) PBS (μmol/kg) Conjugate linkage/Length ID No. PBS 0 100 — — — ISIS 0.08 95 0.77 None PS/20 4878 304801 0.75 42 2.25 32 6.75 19 ISIS 0.08 50 0.074 GalNAc₃-1 PS/20 4879 647535 0.75 15 2.25 17 6.75 8 ApoC III Protein Analysis (Turbidometric Assay)

Plasma ApoC III protein analysis was determined using procedures reported by Graham et al, Circulation Research, published online before print Mar. 29, 2013.

Approximately 100 μl of plasma isolated from mice was analyzed without dilution using an Olympus Clinical Analyzer and a commercially available turbidometric ApoC III assay (Kamiya, Cat #KAI-006, Kamiya Biomedical, Seattle, Wash.). The assay protocol was performed as described by the vendor.

As shown in the Table 19 below, both antisense compounds reduced ApoC III protein relative to the PBS control. Further, the antisense compound conjugated to GalNAc₃-1 (ISIS 647535) was substantially more potent than the antisense compound lacking the GalNAc₃-1 conjugate (ISIS 304801).

TABLE 19 Effect of ASO treatment on ApoC III plasma protein levels in human ApoC III transgenic mice Dose % ED₅₀ 3′ Internucleoside SEQ ASO (μmol/kg) PBS (μmol/kg) Conjugate Linkage/Length ID No. PBS 0 100 — — — ISIS 0.08 86 0.73 None PS/20 4878 304801 0.75 51 2.25 23 6.75 13 ISIS 0.08 72 0.19 GalNAc₃-1 PS/20 4879 647535 0.75 14 2.25 12 6.75 11

Plasma triglycerides and cholesterol were extracted by the method of Bligh and Dyer (Bligh, E. G. and Dyer, W. J. Can. J. Biochem. Physiol. 37: 911-917, 1959)(Bligh, E and Dyer, W, Can J Biochem Physiol, 37, 911-917, 1959)(Bligh, E and Dyer, W, Can J Biochem Physiol, 37, 911-917, 1959) and measured by using a Beckmann Coulter clinical analyzer and commercially available reagents.

The triglyceride levels were measured relative to PBS injected mice and are denoted as “% PBS”. Results are presented in Table 20. As illustrated, both antisense compounds lowered triglyceride levels. Further, the antisense compound conjugated to GalNAc₃-1 (ISIS 647535) was substantially more potent than the antisense compound lacking the GalNAc₃-1 conjugate (ISIS 304801).

TABLE 20 Effect of ASO treatment on triglyceride levels in transgenic mice Dose % ED₅₀ 3′ Internucleoside SEQ ASO (μmol/kg) PBS (μmol/kg) Conjugate Linkage/Length ID No. PBS 0 100 — — — ISIS 0.08 87 0.63 None PS/20 4878 304801 0.75 46 2.25 21 6.75 12 ISIS 0.08 65 0.13 GalNAc₃-1 PS/20 4879 647535 0.75 9 2.25 8 6.75 9

Plasma samples were analyzed by HPLC to determine the amount of total cholesterol and of different fractions of cholesterol (HDL and LDL). Results are presented in Tables 21 and 22. As illustrated, both antisense compounds lowered total cholesterol levels; both lowered LDL; and both raised HDL. Further, the antisense compound conjugated to GalNAc₃-1 (ISIS 647535) was substantially more potent than the antisense compound lacking the GalNAc₃-1 conjugate (ISIS 304801). An increase in HDL and a decrease in LDL levels is a cardiovascular beneficial effect of antisense inhibition of ApoC III.

TABLE 21 Effect of ASO treatment on total cholesterol levels in transgenic mice Total SEQ Dose Cholesterol 3′ Internucleoside ID ASO (μmol/kg) (mg/dL) Conjugate Linkage/Length No. PBS 0 257 — — ISIS 0.08 226 None PS/20 4878 304801 0.75 164 2.25 110 6.75 82 ISIS 0.08 230 GalNAc ₃ -1 PS/20 4879 647535 0.75 82 2.25 86 6.75 99

TABLE 22 Effect of ASO treatment on HDL and LDL cholesterol levels in transgenic mice Dose HDL LDL 3′ Internucleoside SEQ ASO (μmol/kg) (mg/dL) (mg/dL) Conjugate Linkage/Length ID No. PBS 0 17 28 — — ISIS 0.08 17 23 None PS/20 4878 304801 0.75 27 12 2.25 50 4 6.75 45 2 ISIS 0.08 21 21 GalNAc₃-1 PS/20 4879 647535 0.75 44 2 2.25 50 2 6.75 58 2 Pharmacokinetics Analysis (PK)

The PK of the ASOs was also evaluated. Liver and kidney samples were minced and extracted using standard protocols. Samples were analyzed on MSD1 utilizing IP-HPLC-MS. The tissue level (μg/g) of full-length ISIS 304801 and 647535 was measured and the results are provided in Table 23. As illustrated, liver concentrations of total full-length antisense compounds were similar for the two antisense compounds. Thus, even though the GalNAc₃-1-conjugated antisense compound is more active in the liver (as demonstrated by the RNA and protein data above), it is not present at substantially higher concentration in the liver. Indeed, the calculated EC₅₀ (provided in Table 23) confirms that the observed increase in potency of the conjugated compound cannot be entirely attributed to increased accumulation. This result suggests that the conjugate improved potency by a mechanism other than liver accumulation alone, possibly by improving the productive uptake of the antisense compound into cells.

The results also show that the concentration of GalNAc₃-1 conjugated antisense compound in the kidney is lower than that of antisense compound lacking the GalNAc conjugate. This has several beneficial therapeutic implications. For therapeutic indications where activity in the kidney is not sought, exposure to kidney risks kidney toxicity without corresponding benefit. Moreover, high concentration in kidney typically results in loss of compound to the urine resulting in faster clearance. Accordingly, for non-kidney targets, kidney accumulation is undesired. These data suggest that GalNAc₃-1 conjugation reduces kidney accumulation.

TABLE 23 PK analysis of ASO treatment in transgenic mice Dose Liver Kidney Liver EC₅₀ 3′ Internucleoside SEQ ASO (μmol/kg) (μg/g) (μg/g) (μg/g) Conjugate Linkage/Length ID No. ISIS 0.1 5.2 2.1 53 None PS/20 4878 304801 0.8 62.8 119.6 2.3 142.3 191.5 6.8 202.3 337.7 ISIS 0.1 3.8 0.7 3.8 GalNAc₃-1 PS/20 4879 647535 0.8 72.7 34.3 2.3 106.8 111.4 6.8 237.2 179.3

Metabolites of ISIS 647535 were also identified and their masses were confirmed by high resolution mass spectrometry analysis. The cleavage sites and structures of the observed metabolites are shown below. The relative % of full length ASO was calculated using standard procedures and the results are presented in Table 23a. The major metabolite of ISIS 647535 was full-length ASO lacking the entire conjugate (i.e. ISIS 304801), which results from cleavage at cleavage site A, shown below. Further, additional metabolites resulting from other cleavage sites were also observed. These results suggest that introducing other cleabable bonds such as esters, peptides, disulfides, phosphoramidates or acyl-hydrazones between the GalNAc₃-1 sugar and the ASO, which can be cleaved by enzymes inside the cell, or which may cleave in the reductive environment of the cytosol, or which are labile to the acidic pH inside endosomes and lyzosomes, can also be useful.

TABLE 23a Observed full length metabolites of ISIS 647535 Metabolite ASO Cleavage site Relative % 1 ISIS 304801 A 36.1 2 ISIS 304801 + dA B 10.5 3 ISIS 647535 minus [3 GalNAc] C 16.1 4 ISIS 647535 minus D 17.6 [3 GalNAc + 1 5-hydroxy-pentanoic acid tether] 5 ISIS 647535 minus D 9.9 [2 GalNAc + 2 5-hydroxy-pentanoic acid tether] 6 ISIS 647535 minus D 9.8 [3 GalNAc + 3 5-hydroxy-pentanoic acid tether]

 

 

 

 

 

 

Example 21: Antisense Inhibition of Human ApoC III in Human ApoC III Transgenic Mice in Single Administration Study

ISIS 304801, 647535 and 647536 each targeting human ApoC III and described in Table 17, were further evaluated in a single administration study for their ability to inhibit human ApoC III in human ApoC III transgenic mice.

Treatment

Human ApoCIII transgenic mice were maintained on a 12-hour light/dark cycle and fed ad libitum Teklad lab chow. Animals were acclimated for at least 7 days in the research facility before initiation of the experiment. ASOs were prepared in PBS and sterilized by filtering through a 0.2 micron filter. ASOs were dissolved in 0.9% PBS for injection.

Human ApoC III transgenic mice were injected intraperitoneally once at the dosage shown below with ISIS 304801, 647535 or 647536 (described above) or with PBS treated control. The treatment group consisted of 3 animals and the control group consisted of 4 animals. Prior to the treatment as well as after the last dose, blood was drawn from each mouse and plasma samples were analyzed. The mice were sacrificed 72 hours following the last administration.

Samples were collected and analyzed to determine the ApoC III mRNA and protein levels in the liver; plasma triglycerides; and cholesterol, including HDL and LDL fractions were assessed as described above (Example 20). Data from those analyses are presented in Tables 24-28, below. Liver transaminase levels, alanine aminotransferase (ALT) and aspartate aminotransferase (AST), in serum were measured relative to saline injected mice using standard protocols. The ALT and AST levels showed that the antisense compounds were well tolerated at all administered doses.

These results show improvement in potency for antisense compounds comprising a GalNAc₃-1 conjugate at the 3′ terminus (ISIS 647535 and 647536) compared to the antisense compound lacking a GalNAc₃-1 conjugate (ISIS 304801). Further, ISIS 647536, which comprises a GalNAc₃-1 conjugate and some phosphodiester linkages was as potent as ISIS 647535, which comprises the same conjugate and all internucleoside linkages within the ASO are phosphorothioate.

TABLE 24 Effect of ASO treatment on ApoC III mRNA levels in human ApoC III transgenic mice Dose ED₅₀ 3′ Internucleoside SEQ ASO (mg/kg) % PBS (mg/kg) Conjugate linkage/Length ID No. PBS 0 99 — — — ISIS 1 104 13.2 None PS/20 4878 304801 3 92 10 71 30 40 ISIS 0.3 98 1.9 GalNAc₃-1 PS/20 4879 647535 1 70 3 33 10 20 ISIS 0.3 103 1.7 GalNAc₃-1 PS/PO/20 4879 647536 1 60 3 31 10 21

TABLE 25 Effect of ASO treatment on ApoC III plasma protein levels in human ApoC III transgenic mice Dose ED₅₀ 3′ Internucleoside SEQ ASO (mg/kg) % PBS (mg/kg) Conjugate Linkage/Length ID No. PBS 0 99 — — — ISIS 1 104 23.2 None PS/20 4878 304801 3 92 10 71 30 40 ISIS 0.3 98 2.1 GalNAc₃-1 PS/20 4879 647535 1 70 3 33 10 20 ISIS 0.3 103 1.8 GalNAc₃-1 PS/PO/20 4879 647536 1 60 3 31 10 21

TABLE 26 Effect of ASO treatment on triglyceride levels in transgenic mice Dose ED₅₀ 3′ Internucleoside SEQ ASO (mg/kg) % PBS (mg/kg) Conjugate Linkage/Length ID No. PBS 0 98 — — — ISIS 1 80 29.1 None PS/20 4878 304801 3 92 10 70 30 47 ISIS 0.3 100 2.2 GalNAc₃-1 PS/20 4879 647535 1 70 3 34 10 23 ISIS 0.3 95 1.9 GalNAc₃-1 PS/PO/20 4879 647536 1 66 3 31 10 23

TABLE 27 Effect of ASO treatment on total cholesterol levels in transgenic mice Dose 3′ Internucleoside SEQ ASO (mg/kg) % PBS Conjugate Linkage/Length ID No. PBS 0 96 — — ISIS 1 104 None PS/20 4878 304801 3 96 10 86 30 72 ISIS 0.3 93 GalNAc₃-1 PS/20 4879 647535 1 85 3 61 10 53 ISIS 0.3 115 GalNAc₃-1 PS/PO/20 4879 647536 1 79 3 51 10 54

TABLE 28 Effect of ASO treatment on HDL and LDL cholesterol levels in transgenic mice Dose HDL LDL 3′ Internucleoside SEQ ASO (mg/kg) % PBS % PBS Conjugate Linkage/Length ID No. PBS 0 131 90 — — ISIS 1 130 72 None PS/20 4878 304801 3 186 79 10 226 63 30 240 46 ISIS 0.3 98 86 GalNAc₃-1 PS/20 4879 647535 1 214 67 3 212 39 10 218 35 ISIS 0.3 143 89 GalNAc₃-1 PS/PO/20 4879 647536 1 187 56 3 213 33 10 221 34

These results confirm that the GalNAc₃-1 conjugate improves potency of an antisense compound. The results also show equal potency of a GalNAc₃-1 conjugated antisense compounds where the antisense oligonucleotides have mixed linkages (ISIS 647536 which has six phosphodiester linkages) and a full phosphorothioate version of the same antisense compound (ISIS 647535).

Phosphorothioate linkages provide several properties to antisense compounds. For example, they resist nuclease digestion and they bind proteins resulting in accumulation of compound in the liver, rather than in the kidney/urine. These are desirable properties, particularly when treating an indication in the liver. However, phosphorothioate linkages have also been associated with an inflammatory response. Accordingly, reducing the number of phosphorothioate linkages in a compound is expected to reduce the risk of inflammation, but also lower concentration of the compound in liver, increase concentration in the kidney and urine, decrease stability in the presence of nucleases, and lower overall potency. The present results show that a GalNAc₃-1 conjugated antisense compound where certain phosphorothioate linkages have been replaced with phosphodiester linkages is as potent against a target in the liver as a counterpart having full phosphorothioate linkages. Such compounds are expected to be less proinflammatory (See Example 24 describing an experiment showing reduction of PS results in reduced inflammatory effect).

Example 22: Effect of GalNAc₃-1 Conjugated Modified ASO Targeting SRB-1 In Vivo

ISIS 440762 and 651900, each targeting SRB-1 and described in Table 17, were evaluated in a dose-dependent study for their ability to inhibit SRB-1 in Balb/c mice.

Treatment

Six week old male Balb/c mice (Jackson Laboratory, Bar Harbor, Me.) were injected subcutaneously once at the dosage shown below with ISIS 440762, 651900 or with PBS treated control. Each treatment group consisted of 4 animals. The mice were sacrificed 48 hours following the final administration to determine the SRB-1 mRNA levels in liver using real-time PCR and RIBOGREEN® RNA quantification reagent (Molecular Probes, Inc. Eugene, Oreg.) according to standard protocols. SRB-1 mRNA levels were determined relative to total RNA (using Ribogreen), prior to normalization to PBS-treated control. The results below are presented as the average percent of SRB-1 mRNA levels for each treatment group, normalized to PBS-treated control and is denoted as “% PBS”.

As illustrated in Table 29, both antisense compounds lowered SRB-1 mRNA levels. Further, the antisense compound comprising the GalNAc₃-1 conjugate (ISIS 651900) was substantially more potent than the antisense compound lacking the GalNAc₃-1 conjugate (ISIS 440762). These results demonstrate that the potency benefit of GalNAc₃-1 conjugates are observed using antisense oligonucleotides complementary to a different target and having different chemically modified nucleosides, in this instance modified nucleosides comprise constrained ethyl sugar moieties (a bicyclic sugar moiety).

TABLE 29 Effect of ASO treatment on SRB-1 mRNA levels in Balb/c mice Liver SEQ Dose % ED₅₀ 3′ Internucleoside ID ASO (mg/kg) PBS (mg/kg) Conjugate linkage/Length No. PBS 0 100 — — ISIS 0.7 85 2.2 None PS/14 4880 440762 2 55 7 12 20 3 ISIS 0.07 98 0.3 GalNAc₃-1 PS/14 4881 651900 0.2 63 0.7 20 2 6 7 5

Example 23: Human Peripheral Blood Mononuclear Cells (hPBMC) Assay Protocol

The hPBMC assay was performed using BD Vautainer CPT tube method. A sample of whole blood from volunteered donors with informed consent at US HealthWorks clinic (Faraday & El Camino Real, Carlsbad) was obtained and collected in 4-15 BD Vacutainer CPT 8 ml tubes (VWR Cat. #BD362753). The approximate starting total whole blood volume in the CPT tubes for each donor was recorded using the PBMC assay data sheet.

The blood sample was remixed immediately prior to centrifugation by gently inverting tubes 8-10 times. CPT tubes were centrifuged at rt (18-25° C.) in a horizontal (swing-out) rotor for 30 min. at 1500-1800 RCF with brake off (2700 RPM Beckman Allegra 6R). The cells were retrieved from the buffy coat interface (between Ficoll and polymer gel layers); transferred to a sterile 50 ml conical tube and pooled up to 5 CPT tubes/50 ml conical tube/donor. The cells were then washed twice with PBS (Ca⁺⁺, Mg⁺⁺ free; GIBCO). The tubes were topped up to 50 ml and mixed by inverting several times. The sample was then centrifuged at 330×g for 15 minutes at rt (1215 RPM in Beckman Allegra 6R) and aspirated as much supernatant as possible without disturbing pellet. The cell pellet was dislodged by gently swirling tube and resuspended cells in RPMI+10% FBS+pen/strep (˜1 ml/10 ml starting whole blood volume). A 60 μl sample was pipette into a sample vial (Beckman Coulter) with 600 μl VersaLyse reagent (Beckman Coulter Cat #A09777) and was gently vortexed for 10-15 sec. The sample was allowed to incubate for 10 min. at rt and being mixed again before counting. The cell suspension was counted on Vicell XR cell viability analyzer (Beckman Coulter) using PBMC cell type (dilution factor of 1:11 was stored with other parameters). The live cell/ml and viability were recorded. The cell suspension was diluted to 1×10⁷ live PBMC/ml in RPMI+10% FBS+pen/strep.

The cells were plated at 5×10⁵ in 50 l/well of 96-well tissue culture plate (Falcon Microtest). 50 μl/well of 2× concentration oligos/controls diluted in RPMI+10% FBS+pen/strep. was added according to experiment template (100 μl/well total). Plates were placed on the shaker and allowed to mix for approx. 1 min. After being incubated for 24 hrs at 37° C.; 5% CO₂, the plates were centrifuged at 400×g for 10 minutes before removing the supernatant for MSD cytokine assay (i.e. human IL-6, IL-10, IL-8 and MCP-1).

Example 24: Evaluation of Proinflammatory Effects in hPBMC Assay for GalNAc₃-1 Conjugated ASOs

The antisense oligonucleotides (ASOs) listed in Table 30 were evaluated for proinflammatory effect in hPBMC assay using the protocol described in Example 23. ISIS 353512 is an internal standard known to be a high responder for IL-6 release in the assay. The hPBMCs were isolated from fresh, volunteered donors and were treated with ASOs at 0, 0.0128, 0.064, 0.32, 1.6, 8, 40 and 200 μM concentrations. After a 24 hr treatment, the cytokine levels were measured.

The levels of IL-6 were used as the primary readout. The EC₅₀ and E_(max) was calculated using standard procedures. Results are expressed as the average ratio of E_(max)/EC₅₀ from two donors and is denoted as “E_(max)/EC₅₀.” The lower ratio indicates a relative decrease in the proinflammatory response and the higher ratio indicates a relative increase in the proinflammatory response.

With regard to the test compounds, the least proinflammatory compound was the PS/PO linked ASO (ISIS 616468). The GalNAc₃-1 conjugated ASO, ISIS 647535 was slightly less proinflammatory than its non-conjugated counterpart ISIS 304801. These results indicate that incorporation of some PO linkages reduces proinflammatory reaction and addition of a GalNAc₃-1 conjugate does not make a compound more proinflammatory and may reduce proinflammatory response. Accordingly, one would expect that an antisense compound comprising both mixed PS/PO linkages and a GalNAc₃-1 conjugate would produce lower proinflammatory responses relative to full PS linked antisense compound with or without a GalNAc₃-1 conjugate. These results show that GalNAc₃-1 conjugated antisense compounds, particularly those having reduced PS content are less proinflammatory.

Together, these results suggest that a GalNAc₃-1 conjugated compound, particularly one with reduced PS content, can be administered at a higher dose than a counterpart full PS antisense compound lacking a GalNAc₃-1 conjugate. Since half-life is not expected to be substantially different for these compounds, such higher administration would result in less frequent dosing. Indeed such administration could be even less frequent, because the GalNAc₃-1 conjugated compounds are more potent (See Examples 20-22) and re-dosing is necessary once the concentration of a compound has dropped below a desired level, where such desired level is based on potency.

TABLE 30 Modified ASOs SEQ ID ASO Sequence (5′ to 3′) Target No. ISIS G_(es) ^(m)C_(es)T_(es)G_(es)A_(es)T_(ds)T_(ds)A_(ds)G_(ds)A_(ds)G_(ds) TNFα 4882 104838 A_(ds)G_(ds)A_(ds)G_(ds)G_(es)T_(es) ^(m)C_(es) ^(m)C_(es) ^(m)C_(e) ISIS T_(es) ^(m)C_(es) ^(m)C_(es) ^(m)C_(ds)A_(ds)T_(ds)T_(ds)T_(ds) ^(m)C_(ds)A_(ds)G_(ds) CRP 4883 353512 G_(ds)A_(ds)G_(ds)A_(ds) ^(m)C_(ds) ^(m)C_(ds)T_(es)G_(es)G_(e) ISIS A_(es)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(ds)T_(ds)T_(ds)G_(ds)T_(ds) ApoC III 4878 304801 ^(m)C_(ds) ^(m)C_(ds)A_(ds)G_(ds) ^(m)C_(ds)T_(es)T_(es)T_(es)A_(es)T_(e) ISIS A_(es)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(ds)T_(ds)T_(ds)G_(ds)T_(ds) ApoC III 4879 647535 ^(m)C_(ds) ^(m)C_(ds)A_(ds)G_(ds) ^(m)C_(ds)T_(es)T_(es)T_(es)A_(es)T_(eo) A _(do′) -GalNAc ₃ -1 _(a) ISIS A_(es)G_(eo) ^(m)C_(eo)T_(eo)T_(eo) ^(m)C_(ds)T_(ds)T_(ds)G_(ds)T_(ds) ApoC III 4878 616468 ^(m)C_(ds) ^(m)C_(ds)A_(ds)G_(ds) ^(m)C_(ds)T_(eo)T_(eo)T_(es)A_(es)T_(e) Subscripts: “e” indicates 2′-MOE modified nucleoside; “d” indicates β-D-2′-deoxyribonucleoside; “k” indicates 6′-(S)—CH₃ bicyclic nucleoside (e.g. cEt); “s” indicates phosphorothioate internucleoside linkages (PS); “o” indicates phosphodiester internucleoside linkages (PO); and “o′” indicates —O—P(═O)(OH)—. Superscript “m” indicates 5-methylcytosines. “A_(do′)-GalNAc₃-1_(a)” indicates a conjugate having the structure GalNAc₃-1 shown in Example 9 attached to the 3′-end of the antisense oligonucleotide, as indicated.

TABLE 31 Proinflammatory Effect of ASOs targeting ApoC III in hPBMC assay SEQ EC₅₀ E_(max) 3′ Internucleoside ID ASO (μM) (μM) E_(max)/EC₅₀ Conjugate Linkage/Length No. ISIS 0.01 265.9 26,590 None PS/20 4883 353512 (high responder) ISIS 0.07 106.55 1,522 None PS/20 4878 304801 ISIS 0.12 138 1,150 GalNAc₃-1 PS/20 4879 647535 ISIS 0.32 71.52 224 None PS/PO/20 4878 616468

Example 25: Effect of GalNAc₃-1 Conjugated Modified ASO Targeting Human ApoC III In Vitro

ISIS 304801 and 647535 described above were tested in vitro. Primary hepatocyte cells from transgenic mice at a density of 25,000 cells per well were treated with 0.03, 0.08, 0.24, 0.74, 2.22, 6.67 and 20 μM concentrations of modified oligonucleotides. After a treatment period of approximately 16 hours, RNA was isolated from the cells and mRNA levels were measured by quantitative real-time PCR and the hApoC III mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN.

The IC₅₀ was calculated using the standard methods and the results are presented in Table 32. As illustrated, comparable potency was observed in cells treated with ISIS 647535 as compared to the control, ISIS 304801.

TABLE 32 Modified ASO targeting human ApoC III in primary hepatocytes Internucleoside SEQ ASO IC₅₀ (μM) 3′ Conjugate linkage/Length ID No. ISIS 0.44 None PS/20 4878 304801 ISIS 0.31 GalNAc ₃ -1 PS/20 4879 647535

In this experiment, the large potency benefits of GalNAc₃-1 conjugation that are observed in vivo were not observed in vitro. Subsequent free uptake experiments in primary hepatocytes in vitro did show increased potency of oligonucleotides comprising various GalNAc conjugates relative to oligonucleotides that lacking the GalNAc conjugate. (see Examples 60, 82, and 92)

Example 26: Effect of PO/PS Linkages on ApoC III ASO Activity

Human ApoC III transgenic mice were injected intraperitoneally once at 25 mg/kg of ISIS 304801, or ISIS 616468 (both described above) or with PBS treated control once per week for two weeks. The treatment group consisted of 3 animals and the control group consisted of 4 animals. Prior to the treatment as well as after the last dose, blood was drawn from each mouse and plasma samples were analyzed. The mice were sacrificed 72 hours following the last administration.

Samples were collected and analyzed to determine the ApoC III protein levels in the liver as described above (Example 20). Data from those analyses are presented in Table 33, below.

These results show reduction in potency for antisense compounds with PO/PS (ISIS 616468) in the wings relative to full PS (ISIS 304801).

TABLE 33 Effect of ASO treatment on ApoC III protein levels in human ApoC III transgenic mice Dose 3′ Internucleoside SEQ ID ASO (mg/kg) % PBS Conjugate linkage/Length No. PBS 0 99 — — ISIS 25 mg/kg/wk 24 None Full PS 4878 304801 for 2 wks ISIS 25 mg/kg/wk 40 None 14 PS/6 PO 4878 616468 for 2 wks

Example 27: Compound 56

Compound 56 is commercially available from Glen Research or may be prepared according to published procedures reported by Shchepinov et al., Nucleic Acids Research, 1997, 25(22), 4447-4454.

Example 28: Preparation of Compound 60

Compound 4 was prepared as per the procedures illustrated in Example 2. Compound 57 is commercially available. Compound 60 was confirmed by structural analysis.

Compound 57 is meant to be representative and not intended to be limiting as other monoprotected substituted or unsubstituted alkyl diols including but not limited to those presented in the specification herein can be used to prepare phosphoramidites having a predetermined composition.

Example 29: Preparation of Compound 63

Compounds 61 and 62 are prepared using procedures similar to those reported by Tober et al., Eur. J Org. Chem., 2013, 3, 566-577; and Jiang et al., Tetrahedron, 2007, 63(19), 3982-3988.

Alternatively, Compound 63 is prepared using procedures similar to those reported in scientific and patent literature by Kim et al., Synlett, 2003, 12, 1838-1840; and Kim et al., published PCT International Application, WO 2004063208.

Example 30: Preparation of Compound 63b

Compound 63a is prepared using procedures similar to those reported by Hanessian et al., Canadian Journal of Chemistry, 1996, 74(9), 1731-1737.

Example 31: Preparation of Compound 63d

Compound 63c is prepared using procedures similar to those reported by Chen et al., Chinese Chemical Letters, 1998, 9(5), 451-453.

Example 32: Preparation of Compound 67

Compound 64 was prepared as per the procedures illustrated in Example 2. Compound 65 is prepared using procedures similar to those reported by Or et al., published PCT International Application, WO 2009/003009. The protecting groups used for Compound 65 are meant to be representative and not intended to be limiting as other protecting groups including but not limited to those presented in the specification herein can be used.

Example 33: Preparation of Compound 70

Compound 64 was prepared as per the procedures illustrated in Example 2. Compound 68 is commercially available. The protecting group used for Compound 68 is meant to be representative and not intended to be limiting as other protecting groups including but not limited to those presented in the specification herein can be used.

Example 34: Preparation of Compound 75a

Compound 75 is prepared according to published procedures reported by Shchepinov et al., Nucleic Acids Research, 1997, 25(22), 4447-4454.

Example 35: Preparation of Compound 79

Compound 76 was prepared according to published procedures reported by Shchepinov et al., Nucleic Acids Research, 1997, 25(22), 4447-4454.

Example 36: Preparation of Compound 79a

Compound 77 is prepared as per the procedures illustrated in Example 35.

Example 37: General Method for the Preparation of Conjugated Oligomeric Compound 82 Comprising a Phosphodiester Linked GalNAc₃-2 Conjugate at 5′ Terminus Via Solid Support (Method I)

wherein GalNAc₃-2 has the structure:

The GalNAc₃ cluster portion of the conjugate group GalNAc₃-2 (GalNAc₃-2_(a)) can be combined with any cleavable moiety to provide a variety of conjugate groups. Wherein GalNAc₃-2_(a) has the formula:

The VIMAD-bound oligomeric compound 79b was prepared using standard procedures for automated DNA/RNA synthesis (see Dupouy et al., Angew. Chem. Int. Ed., 2006, 45, 3623-3627). The phosphoramidite Compounds 56 and 60 were prepared as per the procedures illustrated in Examples 27 and 28, respectively. The phosphoramidites illustrated are meant to be representative and not intended to be limiting as other phosphoramidite building blocks including but not limited those presented in the specification herein can be used to prepare an oligomeric compound having a phosphodiester linked conjugate group at the 5′ terminus. The order and quantity of phosphoramidites added to the solid support can be adjusted to prepare the oligomeric compounds as described herein having any predetermined sequence and composition.

Example 38: Alternative Method for the Preparation of Oligomeric Compound 82 Comprising a Phosphodiester Linked GalNAc₃-2 Conjugate at 5′ Terminus (Method II)

The VIMAD-bound oligomeric compound 79b was prepared using standard procedures for automated DNA/RNA synthesis (see Dupouy et al., Angew. Chem. Int. Ed., 2006, 45, 3623-3627). The GalNAc₃-2 cluster phosphoramidite, Compound 79 was prepared as per the procedures illustrated in Example 35. This alternative method allows a one-step installation of the phosphodiester linked GalNAc₃-2 conjugate to the oligomeric compound at the final step of the synthesis. The phosphoramidites illustrated are meant to be representative and not intended to be limiting, as other phosphoramidite building blocks including but not limited to those presented in the specification herein can be used to prepare oligomeric compounds having a phosphodiester conjugate at the 5′ terminus. The order and quantity of phosphoramidites added to the solid support can be adjusted to prepare the oligomeric compounds as described herein having any predetermined sequence and composition.

Example 39: General Method for the Preparation of Oligomeric Compound 83h Comprising a GalNAc₃-3 Conjugate at the 5′ Terminus (GalNAc₃-1 Modified for 5′ End Attachment) Via Solid Support

Compound 18 was prepared as per the procedures illustrated in Example 4. Compounds 83a and 83b are commercially available. Oligomeric Compound 83e comprising a phosphodiester linked hexylamine was prepared using standard oligonucleotide synthesis procedures. Treatment of the protected oligomeric compound with aqueous ammonia provided the 5′-GalNAc₃-3 conjugated oligomeric compound (83h).

Wherein GalNAc₃-3 has the structure:

The GalNAc₃ cluster portion of the conjugate group GalNAc₃-3 (GalNAc₃-3_(a)) can be combined with any cleavable moiety to provide a variety of conjugate groups. Wherein GalNAc₃-3_(a) has the formula:

Example 40: General Method for the Preparation of Oligomeric Compound 89 Comprising a Phosphodiester Linked GalNAc₃-4 Conjugate at the 3′ Terminus Via Solid Support

Wherein GalNAc₃-4 has the structure:

Wherein CM is a cleavable moiety. In certain embodiments, cleavable moiety is:

The GalNAc₃ cluster portion of the conjugate group GalNAc₃-4 (GalNAc₃-4_(a)) can be combined with any cleavable moiety to provide a variety of conjugate groups. Wherein GalNAc₃-4_(a) has the formula:

The protected Unylinker functionalized solid support Compound 30 is commercially available. Compound 84 is prepared using procedures similar to those reported in the literature (see Shchepinov et al., Nucleic Acids Research, 1997, 25(22), 4447-4454; Shchepinov et al., Nucleic Acids Research, 1999, 27, 3035-3041; and Hornet et al., Nucleic Acids Research, 1997, 25, 4842-4849).

The phosphoramidite building blocks, Compounds 60 and 79a are prepared as per the procedures illustrated in Examples 28 and 36. The phosphoramidites illustrated are meant to be representative and not intended to be limiting as other phosphoramidite building blocks can be used to prepare an oligomeric compound having a phosphodiester linked conjugate at the 3′ terminus with a predetermined sequence and composition. The order and quantity of phosphoramidites added to the solid support can be adjusted to prepare the oligomeric compounds as described herein having any predetermined sequence and composition.

Example 41: General Method for the Preparation of ASOs Comprising a Phosphodiester Linked GalNAc₃-2 (See Example 37, Bx is Adenine) Conjugate at the 5′ Position Via Solid Phase Techniques (Preparation of ISIS 661134)

Unless otherwise stated, all reagents and solutions used for the synthesis of oligomeric compounds are purchased from commercial sources. Standard phosphoramidite building blocks and solid support are used for incorporation nucleoside residues which include for example T, A, G, and ^(m)C residues. Phosphoramidite compounds 56 and 60 were used to synthesize the phosphodiester linked GalNAc₃-2 conjugate at the 5′ terminus. A 0.1 μM solution of phosphoramidite in anhydrous acetonitrile was used for 3-D-2′-deoxyribonucleoside and 2′-MOE.

The ASO syntheses were performed on ABI 394 synthesizer (1-2 μmol scale) or on GE Healthcare Bioscience ÄKTA oligopilot synthesizer (40-200 μmol scale) by the phosphoramidite coupling method on VIMAD solid support (110 μmol/g, Guzaev et al., 2003) packed in the column. For the coupling step, the phosphoramidites were delivered at a 4 fold excess over the initial loading of the solid support and phosphoramidite coupling was carried out for 10 min. All other steps followed standard protocols supplied by the manufacturer. A solution of 6% dichloroacetic acid in toluene was used for removing the dimethoxytrityl (DMT) groups from 5′-hydroxyl groups of the nucleotide. 4,5-Dicyanoimidazole (0.7 M) in anhydrous CH₃CN was used as activator during the coupling step. Phosphorothioate linkages were introduced by sulfurization with 0.1 μM solution of xanthane hydride in 1:1 pyridine/CH₃CN for a contact time of 3 minutes. A solution of 20% tert-butylhydroperoxide in CH₃CN containing 6% water was used as an oxidizing agent to provide phosphodiester internucleoside linkages with a contact time of 12 minutes.

After the desired sequence was assembled, the cyanoethyl phosphate protecting groups were deprotected using a 20% diethylamine in toluene (v/v) with a contact time of 45 minutes. The solid-support bound ASOs were suspended in aqueous ammonia (28-30 wt %) and heated at 55° C. for 6 h. The unbound ASOs were then filtered and the ammonia was boiled off. The residue was purified by high pressure liquid chromatography on a strong anion exchange column (GE Healthcare Bioscience, Source 30Q, 30 μm, 2.54×8 cm, A=100 mM ammonium acetate in 30% aqueous CH₃CN, B=1.5 μM NaBr in A, 0-40% of B in 60 min, flow 14 mL min-1, =260 nm). The residue was desalted by HPLC on a reverse phase column to yield the desired ASOs in an isolated yield of 15-30% based on the initial loading on the solid support. The ASOs were characterized by ion-pair-HPLC coupled MS analysis with Agilent 1100 MSD system.

TABLE 34 ASO comprising a phosphodiester linked GalNAc₃-2 conjugate at the 5′ position targeting SRB-1 Observed SEQ ID ISIS No. Sequence (5′ to 3′) CalCd Mass Mass No. 661134 GalNAc ₃ -2 _(a) - _(o′) A _(do)T_(ks) ^(m)C_(ks)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds) 6482.2 6481.6 4884 G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(ks) ^(m)C_(k) Subscripts: “e” indicates 2′-MOE modified nucleoside; “d” indicates β-D-2′-deoxyribonucleoside; “k” indicates 6′-(S)—CH₃ bicyclic nucleoside (e.g. cEt); “s” indicates phosphorothioate internucleoside linkages (PS); “o” indicates phosphodiester internucleoside linkages (PO); and “o′” indicates —O—P(═O)(OH)—. Superscript “m” indicates 5-methylcytosines. The structure of GalNAc₃-2_(a) is shown in Example 37.

Example 42: General Method for the Preparation of ASOs Comprising a GalNAc₃-3 Conjugate at the 5′ Position Via Solid Phase Techniques (Preparation of ISIS 661166)

The synthesis for ISIS 661166 was performed using similar procedures as illustrated in Examples 39 and 41.

ISIS 661166 is a 5-10-5 MOE gapmer, wherein the 5′ position comprises a GalNAc₃-3 conjugate. The ASO was characterized by ion-pair-HPLC coupled MS analysis with Agilent 1100 MSD system.

TABLE 34A ASO comprising a GalNAc₃-3 conjugate at the 5′ position via a hexylamino phosphodiester linkage targeting Malat-1 ISIS Calcd Observed No. Sequence (5′ to 3′) Conjugate Mass Mass SEQ ID No. 661166 5′-GalNAc ₃ -3 _(a-o′) ^(m)C_(es)G_(es)G_(es)T_(es)G_(es) 5′ -GalNAc ₃ -3 8992.16 8990.51 4885 ^(m)C_(ds)A_(ds)A_(ds)G_(ds)G_(ds) ^(m)C_(ds)T_(ds)T_(ds)A_(ds)G_(ds) G_(es)A_(es)A_(es)T_(es)T_(e) Subscripts: “e” indicates 2′-MOE modified nucleoside; “d” indicates β-D-2′-deoxyribonucleoside; “s” indicates phosphorothioate internucleoside linkages (PS); “o” indicates phosphodiester internucleoside linkages (PO); and “o′” indicates —O—P(═O)(OH)—. Superscript “m” indicates 5-methylcytosines. The structure of “5′-GalNAc₃-3a” is shown in Example 39.

Example 43: Dose-Dependent Study of Phosphodiester Linked GalNAc₃-2 (See Examples 37 and 41, Bx is Adenine) at the 5′ Terminus Targeting SRB-1 In Vivo

ISIS 661134 (see Example 41) comprising a phosphodiester linked GalNAc₃-2 conjugate at the 5′ terminus was tested in a dose-dependent study for antisense inhibition of SRB-1 in mice. Unconjugated ISIS 440762 and 651900 (GalNAc₃-1 conjugate at 3′ terminus, see Example 9) were included in the study for comparison and are described previously in Table 17.

Treatment

Six week old male Balb/c mice (Jackson Laboratory, Bar Harbor, Me.) were injected subcutaneously once at the dosage shown below with ISIS 440762, 651900, 661134 or with PBS treated control. Each treatment group consisted of 4 animals. The mice were sacrificed 72 hours following the final administration to determine the liver SRB-1 mRNA levels using real-time PCR and RIBOGREEN® RNA quantification reagent (Molecular Probes, Inc. Eugene, Oreg.) according to standard protocols. SRB-1 mRNA levels were determined relative to total RNA (using Ribogreen), prior to normalization to PBS-treated control. The results below are presented as the average percent of SRB-1 mRNA levels for each treatment group, normalized to PBS-treated control and is denoted as “% PBS”. The ED₅₀s were measured using similar methods as described previously and are presented below.

As illustrated in Table 35, treatment with antisense oligonucleotides lowered SRB-1 mRNA levels in a dose-dependent manner. Indeed, the antisense oligonucleotides comprising the phosphodiester linked GalNAc₃-2 conjugate at the 5′ terminus (ISIS 661134) or the GalNAc₃-1 conjugate linked at the 3′ terminus (ISIS 651900) showed substantial improvement in potency compared to the unconjugated antisense oligonucleotide (ISIS 440762). Further, ISIS 661134, which comprises the phosphodiester linked GalNAc₃-2 conjugate at the 5′ terminus was equipotent compared to ISIS 651900, which comprises the GalNAc₃-1 conjugate at the 3′ terminus.

TABLE 35 ASOs containing GalNAc₃-1 or GalNAc₃-2 targeting SRB-1 ISIS Dosage SRB-1 mRNA ED₅₀ SEQ No. (mg/kg) levels (% PBS) (mg/kg) Conjugate ID No. PBS 0 100 — — 440762 0.2 116 2.58 No conjugate 4880 0.7 91 2 69 7 22 20 5 651900 0.07 95 0.26 3′ GalNAc ₃ -1 4881 0.2 77 0.7 28 2 11 7 8 661134 0.07 107 0.25 5′ GalNAc ₃ -2 4881 0.2 86 0.7 28 2 10 7 6

Structures for 3′ GalNAc₃-1 and 5′ GalNAc₃-2 were described previously in Examples 9 and 37.

Pharmacokinetics Analysis (PK)

The PK of the ASOs from the high dose group (7 mg/kg) was examined and evaluated in the same manner as illustrated in Example 20. Liver sample was minced and extracted using standard protocols. The full length metabolites of 661134 (5′ GalNAc₃-2) and ISIS 651900 (3′ GalNAc₃-1) were identified and their masses were confirmed by high resolution mass spectrometry analysis. The results showed that the major metabolite detected for the ASO comprising a phosphodiester linked GalNAc₃-2 conjugate at the 5′ terminus (ISIS 661134) was ISIS 440762 (data not shown). No additional metabolites, at a detectable level, were observed. Unlike its counterpart, additional metabolites similar to those reported previously in Table 23a were observed for the ASO having the GalNAc₃-1 conjugate at the 3′ terminus (ISIS 651900). These results suggest that having the phosphodiester linked GalNAc₃-1 or GalNAc₃-2 conjugate may improve the PK profile of ASOs without compromising their potency.

Example 44: Effect of PO/PS Linkages on Antisense Inhibition of ASOs Comprising GalNAc₃-1 Conjugate (See Example 9) at the 3′ Terminus Targeting SRB-1

ISIS 655861 and 655862 comprising a GalNAc₃-1 conjugate at the 3′ terminus each targeting SRB-1 were tested in a single administration study for their ability to inhibit SRB-1 in mice. The parent unconjugated compound, ISIS 353382 was included in the study for comparison.

The ASOs are 5-10-5 MOE gapmers, wherein the gap region comprises ten 2′-deoxyribonucleosides and each wing region comprises five 2′-MOE modified nucleosides. The ASOs were prepared using similar methods as illustrated previously in Example 19 and are described Table 36, below.

TABLE 36 Modified ASOs comprising GalNAc₃-1 conjugate at the 3′ terminus targeting SRB-1 SEQ ID ISIS No. Sequence (5′ to 3′) Chemistry No. 353382 G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) Full PS no conjugate 4886 (parent) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) 655861 G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) Full PS with 4887 ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(eo) A _(do′) -GalNAc ₃ -1 _(a) GalNAc ₃ -1 conjugate 655862 G_(es) ^(m)C_(eo)T_(eo)T_(eo) ^(m)C_(eo)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) Mixed PS/PO with 4887 ^(m)C_(ds)T_(ds)T_(eo) ^(m)C_(eo) ^(m)C_(es)T_(es)T_(eo) A _(do′) -GalNAc ₃ -1a GalNAc ₃ -1 conjugate Subscripts: “e” indicates 2′-MOE modified nucleoside; “d” indicates β-D-2′-deoxyribonucleoside; “s” indicates phosphorothioate internucleoside linkages (PS); “o” indicates phosphodiester internucleoside linkages (PO); and “o′” indicates —O—P(═O)(OH)—. Superscript “m” indicates 5-methylcytosines. The structure of “GalNAc₃-1” is shown in Example 9. Treatment

Six week old male Balb/c mice (Jackson Laboratory, Bar Harbor, Me.) were injected subcutaneously once at the dosage shown below with ISIS 353382, 655861, 655862 or with PBS treated control. Each treatment group consisted of 4 animals. Prior to the treatment as well as after the last dose, blood was drawn from each mouse and plasma samples were analyzed. The mice were sacrificed 72 hours following the final administration to determine the liver SRB-1 mRNA levels using real-time PCR and RIBOGREEN® RNA quantification reagent (Molecular Probes, Inc. Eugene, Oreg.) according to standard protocols. SRB-1 mRNA levels were determined relative to total RNA (using Ribogreen), prior to normalization to PBS-treated control. The results below are presented as the average percent of SRB-1 mRNA levels for each treatment group, normalized to PBS-treated control and is denoted as “% PBS”. The ED₅₀s were measured using similar methods as described previously and are reported below.

As illustrated in Table 37, treatment with antisense oligonucleotides lowered SRB-1 mRNA levels in a dose-dependent manner compared to PBS treated control. Indeed, the antisense oligonucleotides comprising the GalNAc₃-1 conjugate at the 3′ terminus (ISIS 655861 and 655862) showed substantial improvement in potency comparing to the unconjugated antisense oligonucleotide (ISIS 353382). Further, ISIS 655862 with mixed PS/PO linkages showed an improvement in potency relative to full PS (ISIS 655861).

TABLE 37 Effect of PO/PS linkages on antisense inhibition of ASOs comprising GalNAc₃-1 conjugate at 3′ terminus targeting SRB-1 ISIS Dosage SRB-1 mRNA ED₅₀ SEQ No. (mg/kg) levels (% PBS) (mg/kg) Chemistry ID No. PBS 0 100 — — 353382 3 76.65 10.4 Full PS without 4886 (parent) 10 52.40 conjugate 30 24.95 655861 0.5 81.22 2.2 Full PS with 4887 1.5 63.51 GalNAc₃-1 5 24.61 conjugate 15 14.80 655862 0.5 69.57 1.3 Mixed PS/PO 4887 1.5 45.78 with 5 19.70 GalNAc₃-1 15 12.90 conjugate

Liver transaminase levels, alanine aminotransferase (ALT) and aspartate aminotransferase (AST), in serum were measured relative to saline injected mice using standard protocols. Organ weights were also evaluated. The results demonstrated that no elevation in transaminase levels (Table 38) or organ weights (data not shown) were observed in mice treated with ASOs compared to PBS control. Further, the ASO with mixed PS/PO linkages (ISIS 655862) showed similar transaminase levels compared to full PS (ISIS 655861).

TABLE 38 Effect of PO/PS linkages on transaminase levels of ASOs comprising GalNAc₃-1 conjugate at 3′ terminus targeting SRB-1 Dosage ALT AST SEQ ISIS No. (mg/kg) (U/L) (U/L) Chemistry ID No. PBS 0 28.5 65 — 353382 3 50.25 89 Full PS without 4886 (parent) 10 27.5 79.3 conjugate 30 27.3 97 655861 0.5 28 55.7 Full PS with 4887 1.5 30 78 GalNAc₃-1 5 29 63.5 15 28.8 67.8 655862 0.5 50 75.5 Mixed PS/PO with 4887 1.5 21.7 58.5 GalNAc₃-1 5 29.3 69 15 22 61

Example 45: Preparation of PFP Ester, Compound 110a

Compound 4 (9.5 g, 28.8 mmoles) was treated with compound 103a or 103b (38 mmoles), individually, and TMSOTf (0.5 eq.) and molecular sieves in dichloromethane (200 mL), and stirred for 16 hours at room temperature. At that time, the organic layer was filtered thru celite, then washed with sodium bicarbonate, water and brine. The organic layer was then separated and dried over sodium sulfate, filtered and reduced under reduced pressure. The resultant oil was purified by silica gel chromatography (2%-→10% methanol/dichloromethane) to give compounds 104a and 104b in >80% yield. LCMS and proton NMR was consistent with the structure.

Compounds 104a and 104b were treated to the same conditions as for compounds 100a-d (Example 47), to give compounds 105a and 105b in >90% yield. LCMS and proton NMR was consistent with the structure.

Compounds 105a and 105b were treated, individually, with compound 90 under the same conditions as for compounds 901a-d, to give compounds 106a (80%) and 106b (20%). LCMS and proton NMR was consistent with the structure.

Compounds 106a and 106b were treated to the same conditions as for compounds 96a-d (Example 47), to give 107a (60%) and 107b (20%). LCMS and proton NMR was consistent with the structure.

Compounds 107a and 107b were treated to the same conditions as for compounds 97a-d (Example 47), to give compounds 108a and 108b in 40-60% yield. LCMS and proton NMR was consistent with the structure.

Compounds 108a (60%) and 108b (40%) were treated to the same conditions as for compounds 100a-d (Example 47), to give compounds 109a and 109b in >80% yields. LCMS and proton NMR was consistent with the structure.

Compound 109a was treated to the same conditions as for compounds 101a-d (Example 47), to give Compound 110a in 30-60% yield. LCMS and proton NMR was consistent with the structure. Alternatively, Compound 110b can be prepared in a similar manner starting with Compound 109b.

Example 46: General Procedure for Conjugation with PFP Esters (Oligonucleotide 111); Preparation of ISIS 666881 (GalNAc₃-10)

A 5′-hexylamino modified oligonucleotide was synthesized and purified using standard solid-phase oligonucleotide procedures. The 5′-hexylamino modified oligonucleotide was dissolved in 0.1 μM sodium tetraborate, pH 8.5 (200 μL) and 3 equivalents of a selected PFP esterified GalNAc₃ cluster dissolved in DMSO (50 μL) was added. If the PFP ester precipitated upon addition to the ASO solution DMSO was added until all PFP ester was in solution. The reaction was complete after about 16 h of mixing at room temperature. The resulting solution was diluted with water to 12 mL and then spun down at 3000 rpm in a spin filter with a mass cut off of 3000 Da. This process was repeated twice to remove small molecule impurities. The solution was then lyophilized to dryness and redissolved in concentrated aqueous ammonia and mixed at room temperature for 2.5 h followed by concentration in vacuo to remove most of the ammonia. The conjugated oligonucleotide was purified and desalted by RP-HPLC and lyophilized to provide the GalNAc₃ conjugated oligonucleotide.

Oligonucleotide 111 is conjugated with GalNAc₃-10. The GalNAc₃ cluster portion of the conjugate group GalNAc₃-10 (GalNAc₃-10_(a)) can be combined with any cleavable moiety to provide a variety of conjugate groups. In certain embodiments, the cleavable moiety is —P(═O)(OH)-A_(d)-P(═O)(OH)— as shown in the oligonucleotide (ISIS 666881) synthesized with GalNAc₃-10 below. The structure of GalNAc₃-10 (GalNAc₃-10_(a)-CM-) is shown below:

Following this general procedure ISIS 666881 was prepared. 5′-hexylamino modified oligonucleotide, ISIS 660254, was synthesized and purified using standard solid-phase oligonucleotide procedures. ISIS 660254 (40 mg, 5.2 μmol) was dissolved in 0.1 μM sodium tetraborate, pH 8.5 (200 μL) and 3 equivalents PFP ester (Compound 110a) dissolved in DMSO (50 μL) was added. The PFP ester precipitated upon addition to the ASO solution requiring additional DMSO (600 μL) to fully dissolve the PFP ester. The reaction was complete after 16 h of mixing at room temperature. The solution was diluted with water to 12 mL total volume and spun down at 3000 rpm in a spin filter with a mass cut off of 3000 Da. This process was repeated twice to remove small molecule impurities. The solution was lyophilized to dryness and redissolved in concentrated aqueous ammonia with mixing at room temperature for 2.5 h followed by concentration in vacuo to remove most of the ammonia. The conjugated oligonucleotide was purified and desalted by RP-HPLC and lyophilized to give ISIS 666881 in 90% yield by weight (42 mg, 4.7 μmol).

GalNAc₃-10 conjugated oligonucleotide SEQ ASO Sequence (5′ to 3′) 5′ group ID No. ISIS 660254 NH₂(CH₂)₆-_(o)A_(do)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) Hexylamine 4888 ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(es)T_(ds) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) ISIS 666881 GalNAc ₃ -10 _(a) - _(o′) A _(do)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) GalNAc ₃ -10 4888 ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) Capital letters indicate the nucleobase for each nucleoside and ^(m)C indicates a 5-methyl cytosine. Subscripts: “e” indicates a 2′-MOE modified nucleoside; “d” indicates a β-D-2′-deoxyribonucleoside; “s” indicates a phosphorothioate internucleoside linkage (PS); “o” indicates a phosphodiester internucleoside linkage (PO); and “o′” indicates —O—P(═O)(OH)—. Conjugate groups are in bold.

Example 47: Preparation of Oligonucleotide 102 Comprising GalNAc₃-8

The triacid 90 (4 g, 14.43 mmol) was dissolved in DMF (120 mL) and N,N-Diisopropylethylamine (12.35 mL, 72 mmoles). Pentafluorophenyl trifluoroacetate (8.9 mL, 52 mmoles) was added dropwise, under argon, and the reaction was allowed to stir at room temperature for 30 minutes. Boc-diamine 91a or 91b (68.87 mmol) was added, along with N,N-Diisopropylethylamine (12.35 mL, 72 mmoles), and the reaction was allowed to stir at room temperature for 16 hours. At that time, the DMF was reduced by >75% under reduced pressure, and then the mixture was dissolved in dichloromethane. The organic layer was washed with sodium bicarbonate, water and brine. The organic layer was then separated and dried over sodium sulfate, filtered and reduced to an oil under reduced pressure. The resultant oil was purified by silica gel chromatography (2%-→10% methanol/dichloromethane) to give compounds 92a and 92b in an approximate 80% yield. LCMS and proton NMR were consistent with the structure.

Compound 92a or 92b (6.7 mmoles) was treated with 20 mL of dichloromethane and 20 mL of trifluoroacetic acid at room temperature for 16 hours. The resultant solution was evaporated and then dissolved in methanol and treated with DOWEX-OH resin for 30 minutes. The resultant solution was filtered and reduced to an oil under reduced pressure to give 85-90% yield of compounds 93a and 93b.

Compounds 7 or 64 (9.6 mmoles) were treated with HBTU (3.7 g, 9.6 mmoles) and N,N-Diisopropylethylamine (5 mL) in DMF (20 mL) for 15 minutes. To this was added either compounds 93a or 93b (3 mmoles), and allowed to stir at room temperature for 16 hours. At that time, the DMF was reduced by >75% under reduced pressure, and then the mixture was dissolved in dichloromethane. The organic layer was washed with sodium bicarbonate, water and brine. The organic layer was then separated and dried over sodium sulfate, filtered and reduced to an oil under reduced pressure. The resultant oil was purified by silica gel chromatography (5%-→20% methanol/dichloromethane) to give compounds 96a-d in 20-40% yield. LCMS and proton NMR was consistent with the structure.

Compounds 96a-d (0.75 mmoles), individually, were hydrogenated over Raney Nickel for 3 hours in Ethanol (75 mL). At that time, the catalyst was removed by filtration thru celite, and the ethanol removed under reduced pressure to give compounds 97a-d in 80-90% yield. LCMS and proton NMR were consistent with the structure.

Compound 23 (0.32 g, 0.53 mmoles) was treated with HBTU (0.2 g, 0.53 mmoles) and N,N-Diisopropylethylamine (0.19 mL, 1.14 mmoles) in DMF (30 mL) for 15 minutes. To this was added compounds 97a-d (0.38 mmoles), individually, and allowed to stir at room temperature for 16 hours. At that time, the DMF was reduced by >75% under reduced pressure, and then the mixture was dissolved in dichloromethane. The organic layer was washed with sodium bicarbonate, water and brine. The organic layer was then separated and dried over sodium sulfate, filtered and reduced to an oil under reduced pressure. The resultant oil was purified by silica gel chromatography (2%-→20% methanol/dichloromethane) to give compounds 98a-d in 30-40% yield. LCMS and proton NMR was consistent with the structure.

Compound 99 (0.17 g, 0.76 mmoles) was treated with HBTU (0.29 g, 0.76 mmoles) and N,N-Diisopropylethylamine (0.35 mL, 2.0 mmoles) in DMF (50 mL) for 15 minutes. To this was added compounds 97a-d (0.51 mmoles), individually, and allowed to stir at room temperature for 16 hours. At that time, the DMF was reduced by >75% under reduced pressure, and then the mixture was dissolved in dichloromethane. The organic layer was washed with sodium bicarbonate, water and brine. The organic layer was then separated and dried over sodium sulfate, filtered and reduced to an oil under reduced pressure. The resultant oil was purified by silica gel chromatography (5%-→20% methanol/dichloromethane) to give compounds 100a-d in 40-60% yield. LCMS and proton NMR was consistent with the structure.

Compounds 100a-d (0.16 mmoles), individually, were hydrogenated over 10% Pd(OH)₂/C for 3 hours in methanol/ethyl acetate (1:1, 50 mL). At that time, the catalyst was removed by filtration thru celite, and the organics removed under reduced pressure to give compounds 101a-d in 80-90% yield. LCMS and proton NMR was consistent with the structure.

Compounds 101a-d (0.15 mmoles), individually, were dissolved in DMF (15 mL) and pyridine (0.016 mL, 0.2 mmoles). Pentafluorophenyl trifluoroacetate (0.034 mL, 0.2 mmoles) was added dropwise, under argon, and the reaction was allowed to stir at room temperature for 30 minutes. At that time, the DMF was reduced by >75% under reduced pressure, and then the mixture was dissolved in dichloromethane. The organic layer was washed with sodium bicarbonate, water and brine. The organic layer was then separated and dried over sodium sulfate, filtered and reduced to an oil under reduced pressure. The resultant oil was purified by silica gel chromatography (2%-→5% methanol/dichloromethane) to give compounds 102a-d in an approximate 80% yield. LCMS and proton NMR were consistent with the structure.

Oligomeric Compound 102, comprising a GalNAc₃-8 conjugate group, was prepared using the general procedures illustrated in Example 46. The GalNAc₃ cluster portion of the conjugate group GalNAc₃-8 (GalNAc₃-8_(a)) can be combined with any cleavable moiety to provide a variety of conjugate groups. In a preferred embodiment, the cleavable moiety is —P(═O)(OH)-A_(d)-P(═O)(OH)—.

The structure of GalNAc₃-8 (GalNAc₃-8_(a)-CM-) is shown below:

Example 48: Preparation of Oligonucleotide 119 Comprising GalNAc₃-7

Compound 112 was synthesized following the procedure described in the literature (J. Med. Chem. 2004, 47, 5798-5808).

Compound 112 (5 g, 8.6 mmol) was dissolved in 1:1 methanol/ethyl acetate (22 mL/22 mL). Palladium hydroxide on carbon (0.5 g) was added. The reaction mixture was stirred at room temperature under hydrogen for 12 h. The reaction mixture was filtered through a pad of celite and washed the pad with 1:1 methanol/ethyl acetate. The filtrate and the washings were combined and concentrated to dryness to yield Compound 105a (quantitative). The structure was confirmed by LCMS.

Compound 113 (1.25 g, 2.7 mmol), HBTU (3.2 g, 8.4 mmol) and DIEA (2.8 mL, 16.2 mmol) were dissolved in anhydrous DMF (17 mL) and the reaction mixture was stirred at room temperature for 5 min. To this a solution of Compound 105a (3.77 g, 8.4 mmol) in anhydrous DMF (20 mL) was added. The reaction was stirred at room temperature for 6 h. Solvent was removed under reduced pressure to get an oil. The residue was dissolved in CH₂Cl₂ (100 mL) and washed with aqueous saturated NaHCO₃ solution (100 mL) and brine (100 mL). The organic phase was separated, dried (Na₂SO₄), filtered and evaporated. The residue was purified by silica gel column chromatography and eluted with 10 to 20% MeOH in dichloromethane to yield Compound 114 (1.45 g, 30%). The structure was confirmed by LCMS and ¹H NMR analysis.

Compound 114 (1.43 g, 0.8 mmol) was dissolved in 1:1 methanol/ethyl acetate (4 mL/4 mL). Palladium on carbon (wet, 0.14 g) was added. The reaction mixture was flushed with hydrogen and stirred at room temperature under hydrogen for 12 h. The reaction mixture was filtered through a pad of celite. The celite pad was washed with methanol/ethyl acetate (1:1). The filtrate and the washings were combined together and evaporated under reduced pressure to yield Compound 115 (quantitative). The structure was confirmed by LCMS and ¹H NMR analysis.

Compound 83a (0.17 g, 0.75 mmol), HBTU (0.31 g, 0.83 mmol) and DIEA (0.26 mL, 1.5 mmol) were dissolved in anhydrous DMF (5 mL) and the reaction mixture was stirred at room temperature for 5 min. To this a solution of Compound 115 (1.22 g, 0.75 mmol) in anhydrous DMF was added and the reaction was stirred at room temperature for 6 h. The solvent was removed under reduced pressure and the residue was dissolved in CH₂Cl₂. The organic layer was washed aqueous saturated NaHCO₃ solution and brine and dried over anhydrous Na₂SO₄ and filtered. The organic layer was concentrated to dryness and the residue obtained was purified by silica gel column chromatography and eluted with 3 to 15% MeOH in dichloromethane to yield Compound 116 (0.84 g, 61%). The structure was confirmed by LC MS and ¹H NMR analysis.

Compound 116 (0.74 g, 0.4 mmol) was dissolved in 1:1 methanol/ethyl acetate (5 mL/5 mL). Palladium on carbon (wet, 0.074 g) was added. The reaction mixture was flushed with hydrogen and stirred at room temperature under hydrogen for 12 h. The reaction mixture was filtered through a pad of celite. The celite pad was washed with methanol/ethyl acetate (1:1). The filtrate and the washings were combined together and evaporated under reduced pressure to yield compound 117 (0.73 g, 98%). The structure was confirmed by LCMS and ¹H NMR analysis.

Compound 117 (0.63 g, 0.36 mmol) was dissolved in anhydrous DMF (3 mL). To this solution N,N-Diisopropylethylamine (70 μL, 0.4 mmol) and pentafluorophenyl trifluoroacetate (72 μL, 0.42 mmol) were added. The reaction mixture was stirred at room temperature for 12 h and poured into a aqueous saturated NaHCO₃ solution. The mixture was extracted with dichloromethane, washed with brine and dried over anhydrous Na₂SO₄. The dichloromethane solution was concentrated to dryness and purified with silica gel column chromatography and eluted with 5 to 10% MeOH in dichloromethane to yield compound 118 (0.51 g, 79%). The structure was confirmed by LCMS and ¹H and ¹H and ¹⁹F NMR.

Oligomeric Compound 119, comprising a GalNAc₃-7 conjugate group, was prepared using the general procedures illustrated in Example 46. The GalNAc₃ cluster portion of the conjugate group GalNAc₃-7 (GalNAc₃-7_(a)) can be combined with any cleavable moiety to provide a variety of conjugate groups. In certain embodiments, the cleavable moiety is —P(═O)(OH)-A_(d)-P(═O)(OH)—.

The structure of GalNAc₃-7 (GalNAc₃-7_(a)-CM-) is shown below:

Example 49: Preparation of Oligonucleotide 132 Comprising GalNAc₃-5

Compound 120 (14.01 g, 40 mmol) and HBTU (14.06 g, 37 mmol) were dissolved in anhydrous DMF (80 mL). Triethylamine (11.2 mL, 80.35 mmol) was added and stirred for 5 min. The reaction mixture was cooled in an ice bath and a solution of compound 121 (10 g, mmol) in anhydrous DMF (20 mL) was added. Additional triethylamine (4.5 mL, 32.28 mmol) was added and the reaction mixture was stirred for 18 h under an argon atmosphere. The reaction was monitored by TLC (ethyl acetate:hexane; 1:1; Rf=0.47). The solvent was removed under reduced pressure. The residue was taken up in EtOAc (300 mL) and washed with 1M NaHSO₄ (3×150 mL), aqueous saturated NaHCO₃ solution (3×150 mL) and brine (2×100 mL). Organic layer was dried with Na₂SO₄. Drying agent was removed by filtration and organic layer was concentrated by rotary evaporation. Crude mixture was purified by silica gel column chromatography and eluted by using 35-50% EtOAc in hexane to yield a compound 122 (15.50 g, 78.13%). The structure was confirmed by LCMS and ¹H NMR analysis. Mass m/z 589.3 [M+H]⁺.

A solution of LiOH (92.15 mmol) in water (20 mL) and THF (10 mL) was added to a cooled solution of Compound 122 (7.75 g, 13.16 mmol) dissolved in methanol (15 mL). The reaction mixture was stirred at room temperature for 45 min. and monitored by TLC (EtOAc:hexane; 1:1). The reaction mixture was concentrated to half the volume under reduced pressure. The remaining solution was cooled an ice bath and neutralized by adding concentrated HCl. The reaction mixture was diluted, extracted with EtOAc (120 mL) and washed with brine (100 mL). An emulsion formed and cleared upon standing overnight. The organic layer was separated dried (Na₂SO₄), filtered and evaporated to yield Compound 123 (8.42 g). Residual salt is the likely cause of excess mass. LCMS is consistent with structure. Product was used without any further purification. M.W.cal: 574.36; M.W.fd: 575.3 [M+H]⁺.

Compound 126 was synthesized following the procedure described in the literature (J. Am. Chem. Soc. 2011, 133, 958-963).

Compound 123 (7.419 g, 12.91 mmol), HOBt (3.49 g, 25.82 mmol) and compound 126 (6.33 g, 16.14 mmol) were dissolved in and DMF (40 mL) and the resulting reaction mixture was cooled in an ice bath. To this N,N-Diisopropylethylamine (4.42 mL, 25.82 mmol), PyBop (8.7 g, 16.7 mmol) followed by Bop coupling reagent (1.17 g, 2.66 mmol) were added under an argon atmosphere. The ice bath was removed and the solution was allowed to warm to room temperature. The reaction was completed after 1 h as determined by TLC (DCM:MeOH:AA; 89:10:1). The reaction mixture was concentrated under reduced pressure. The residue was dissolved in EtOAc (200 mL) and washed with 1 μM NaHSO₄ (3×100 mL), aqueous saturated NaHCO₃ (3×100 mL) and brine (2×100 mL). The organic phase separated dried (Na₂SO₄), filtered and concentrated. The residue was purified by silica gel column chromatography with a gradient of 50% hexanes/EtOAC to 100% EtOAc to yield Compound 127 (9.4 g) as a white foam. LCMS and ¹H NMR were consistent with structure. Mass m/z 778.4 [M+H]⁺.

Trifluoroacetic acid (12 mL) was added to a solution of compound 127 (1.57 g, 2.02 mmol) in dichloromethane (12 mL) and stirred at room temperature for 1 h. The reaction mixture was co-evaporated with toluene (30 mL) under reduced pressure to dryness. The residue obtained was co-evaporated twice with acetonitrile (30 mL) and toluene (40 mL) to yield Compound 128 (1.67 g) as trifluoro acetate salt and used for next step without further purification. LCMS and ¹H NMR were consistent with structure. Mass m/z 478.2 [M+H]⁺.

Compound 7 (0.43 g, 0.963 mmol), HATU (0.35 g, 0.91 mmol), and HOAt (0.035 g, 0.26 mmol) were combined together and dried for 4 h over P₂O₅ under reduced pressure in a round bottom flask and then dissolved in anhydrous DMF (1 mL) and stirred for 5 min. To this a solution of compound 128 (0.20 g, 0.26 mmol) in anhydrous DMF (0.2 mL) and N,N-Diisopropylethylamine (0.2 mL) was added. The reaction mixture was stirred at room temperature under an argon atmosphere. The reaction was complete after 30 min as determined by LCMS and TLC (7% MeOH/DCM). The reaction mixture was concentrated under reduced pressure. The residue was dissolved in DCM (30 mL) and washed with 1 μM NaHSO₄ (3×20 mL), aqueous saturated NaHCO₃ (3×20 mL) and brine (3×20 mL). The organic phase was separated, dried over Na₂SO₄, filtered and concentrated. The residue was purified by silica gel column chromatography using 5-15% MeOH in dichloromethane to yield Compound 129 (96.6 mg). LC MS and ¹H NMR are consistent with structure. Mass m/z 883.4 [M+2H]⁺.

Compound 129 (0.09 g, 0.051 mmol) was dissolved in methanol (5 mL) in 20 mL scintillation vial. To this was added a small amount of 10% Pd/C (0.015 mg) and the reaction vessel was flushed with H₂ gas. The reaction mixture was stirred at room temperature under H₂ atmosphere for 18 h. The reaction mixture was filtered through a pad of Celite and the Celite pad was washed with methanol. The filtrate washings were pooled together and concentrated under reduced pressure to yield Compound 130 (0.08 g). LCMS and ¹H NMR were consistent with structure. The product was used without further purification. Mass m/z 838.3 [M+2H]⁺.

To a 10 mL pointed round bottom flask were added compound 130 (75.8 mg, 0.046 mmol), 0.37 M pyridine/DMF (200 μL) and a stir bar. To this solution was added 0.7 μM pentafluorophenyl trifluoroacetate/DMF (100 μL) drop wise with stirring. The reaction was completed after 1 h as determined by LC MS. The solvent was removed under reduced pressure and the residue was dissolved in CHCl₃ (˜10 mL). The organic layer was partitioned against NaHSO₄ (1 μM, 10 mL), aqueous saturated NaHCO₃ (10 mL) and brine (10 mL) three times each. The organic phase separated and dried over Na₂SO₄, filtered and concentrated to yield Compound 131 (77.7 mg). LCMS is consistent with structure. Used without further purification. Mass m/z 921.3 [M+2H]⁺.

Oligomeric Compound 132, comprising a GalNAc₃-5 conjugate group, was prepared using the general procedures illustrated in Example 46. The GalNAc₃ cluster portion of the conjugate group GalNAc₃-5 (GalNAc₃-5_(a)) can be combined with any cleavable moiety to provide a variety of conjugate groups. In certain embodiments, the cleavable moiety is —P(═O)(OH)-A_(d)-P(═O)(OH)—.

The structure of GalNAc₃-5 (GalNAc₃-5_(a)-CM-) is shown below:

Example 50: Preparation of Oligonucleotide 144 Comprising GalNAc₄-11

Synthesis of Compound 134. To a Merrifield flask was added aminomethyl VIMAD resin (2.5 g, 450 mol/g) that was washed with acetonitrile, dimethylformamide, dichloromethane and acetonitrile. The resin was swelled in acetonitrile (4 mL). Compound 133 was pre-activated in a 100 mL round bottom flask by adding 20 (1.0 mmol, 0.747 g), TBTU (1.0 mmol, 0.321 g), acetonitrile (5 mL) and DIEA (3.0 mmol, 0.5 mL). This solution was allowed to stir for 5 min and was then added to the Merrifield flask with shaking. The suspension was allowed to shake for 3 h. The reaction mixture was drained and the resin was washed with acetonitrile, DMF and DCM. New resin loading was quantitated by measuring the absorbance of the DMT cation at 500 nm (extinction coefficient=76000) in DCM and determined to be 238 μmol/g. The resin was capped by suspending in an acetic anhydride solution for ten minutes three times.

The solid support bound compound 141 was synthesized using iterative Fmoc-based solid phase peptide synthesis methods. A small amount of solid support was withdrawn and suspended in aqueous ammonia (28-30 wt %) for 6 h. The cleaved compound was analyzed by LC-MS and the observed mass was consistent with structure. Mass m/z 1063.8 [M+2H]⁺.

The solid support bound compound 142 was synthesized using solid phase peptide synthesis methods.

The solid support bound compound 143 was synthesized using standard solid phase synthesis on a DNA synthesizer.

The solid support bound compound 143 was suspended in aqueous ammonia (28-30 wt %) and heated at 55° C. for 16 h. The solution was cooled and the solid support was filtered. The filtrate was concentrated and the residue dissolved in water and purified by HPLC on a strong anion exchange column. The fractions containing full length compound 144 were pooled together and desalted. The resulting GalNAc₄-11 conjugated oligomeric compound was analyzed by LC-MS and the observed mass was consistent with structure.

The GalNAc₄ cluster portion of the conjugate group GalNAc₄-11 (GalNAc₄-11_(a)) can be combined with any cleavable moiety to provide a variety of conjugate groups. In certain embodiments, the cleavable moiety is —P(═O)(OH)-A_(d)-P(═O)(OH)—.

The structure of GalNAc₄-11 (GalNAc₄-11_(a)-CM) is shown below:

Example 51: Preparation of Oligonucleotide 155 Comprising GalNAc₃-6

Compound 146 was synthesized as described in the literature (Analytical Biochemistry 1995, 229, 54-60).

Compound 4 (15 g, 45.55 mmol) and compound 35b (14.3 grams, 57 mmol) were dissolved in CH₂Cl₂ (200 ml). Activated molecular sieves (4 Å. 2 g, powdered) were added, and the reaction was allowed to stir for 30 minutes under nitrogen atmosphere. TMS-OTf was added (4.1 ml, 22.77 mmol) and the reaction was allowed to stir at room temp overnight. Upon completion, the reaction was quenched by pouring into solution of saturated aqueous NaHCO₃ (500 ml) and crushed ice (˜150 g). The organic layer was separated, washed with brine, dried over MgSO₄, filtered, and was concentrated to an orange oil under reduced pressure. The crude material was purified by silica gel column chromatography and eluted with 2-10% MeOH in CH₂Cl₂ to yield Compound 112 (16.53 g, 63%). LCMS and ¹H NMR were consistent with the expected compound.

Compound 112 (4.27 g, 7.35 mmol) was dissolved in 1:1 MeOH/EtOAc (40 ml). The reaction mixture was purged by bubbling a stream of argon through the solution for 15 minutes. Pearlman's catalyst (palladium hydroxide on carbon, 400 mg) was added, and hydrogen gas was bubbled through the solution for 30 minutes. Upon completion (TLC 10% MeOH in CH₂Cl₂, and LCMS), the catalyst was removed by filtration through a pad of celite. The filtrate was concentrated by rotary evaporation, and was dried briefly under high vacuum to yield Compound 105a (3.28 g). LCMS and 1H NMR were consistent with desired product.

Compound 147 (2.31 g, 11 mmol) was dissolved in anhydrous DMF (100 mL). N,N-Diisopropylethylamine (DIEA, 3.9 mL, 22 mmol) was added, followed by HBTU (4 g, 10.5 mmol). The reaction mixture was allowed to stir for ˜15 minutes under nitrogen. To this a solution of compound 105a (3.3 g, 7.4 mmol) in dry DMF was added and stirred for 2 h under nitrogen atmosphere. The reaction was diluted with EtOAc and washed with saturated aqueous NaHCO₃ and brine. The organics phase was separated, dried (MgSO₄), filtered, and concentrated to an orange syrup. The crude material was purified by column chromatography 2-5% MeOH in CH₂Cl₂ to yield Compound 148 (3.44 g, 73%). LCMS and ¹H NMR were consistent with the expected product.

Compound 148 (3.3 g, 5.2 mmol) was dissolved in 1:1 MeOH/EtOAc (75 ml). The reaction mixture was purged by bubbling a stream of argon through the solution for 15 minutes. Pearlman's catalyst (palladium hydroxide on carbon) was added (350 mg). Hydrogen gas was bubbled through the solution for 30 minutes. Upon completion (TLC 10% MeOH in DCM, and LCMS), the catalyst was removed by filtration through a pad of celite. The filtrate was concentrated by rotary evaporation, and was dried briefly under high vacuum to yield Compound 149 (2.6 g). LCMS was consistent with desired product. The residue was dissolved in dry DMF (10 ml) was used immediately in the next step.

Compound 146 (0.68 g, 1.73 mmol) was dissolved in dry DMF (20 ml). To this DIEA (450 μL, 2.6 mmol, 1.5 eq.) and HBTU (1.96 g, 0.5.2 mmol) were added. The reaction mixture was allowed to stir for 15 minutes at room temperature under nitrogen. A solution of compound 149 (2.6 g) in anhydrous DMF (10 mL) was added. The pH of the reaction was adjusted to pH=9-10 by addition of DIEA (if necessary). The reaction was allowed to stir at room temperature under nitrogen for 2 h. Upon completion the reaction was diluted with EtOAc (100 mL), and washed with aqueous saturated aqueous NaHCO₃, followed by brine. The organic phase was separated, dried over MgSO₄, filtered, and concentrated. The residue was purified by silica gel column chromatography and eluted with 2-10% MeOH in CH₂Cl₂ to yield Compound 150 (0.62 g, 20%). LCMS and ¹H NMR were consistent with the desired product.

Compound 150 (0.62 g) was dissolved in 1:1 MeOH/EtOAc (5 L). The reaction mixture was purged by bubbling a stream of argon through the solution for 15 minutes. Pearlman's catalyst (palladium hydroxide on carbon) was added (60 mg). Hydrogen gas was bubbled through the solution for 30 minutes. Upon completion (TLC 10% MeOH in DCM, and LCMS), the catalyst was removed by filtration (syringe-tip Teflon filter, 0.45 μm). The filtrate was concentrated by rotary evaporation, and was dried briefly under high vacuum to yield Compound 151 (0.57 g). The LCMS was consistent with the desired product. The product was dissolved in 4 mL dry DMF and was used immediately in the next step.

Compound 83a (0.11 g, 0.33 mmol) was dissolved in anhydrous DMF (5 mL) and N,N-Diisopropylethylamine (75 μL, 1 mmol) and PFP-TFA (90 μL, 0.76 mmol) were added. The reaction mixture turned magenta upon contact, and gradually turned orange over the next 30 minutes. Progress of reaction was monitored by TLC and LCMS. Upon completion (formation of the PFP ester), a solution of compound 151 (0.57 g, 0.33 mmol) in DMF was added. The pH of the reaction was adjusted to pH=9-10 by addition of N,N-Diisopropylethylamine (if necessary). The reaction mixture was stirred under nitrogen for ˜30 min. Upon completion, the majority of the solvent was removed under reduced pressure. The residue was diluted with CH₂Cl₂ and washed with aqueous saturated NaHCO₃, followed by brine. The organic phase separated, dried over MgSO₄, filtered, and concentrated to an orange syrup. The residue was purified by silica gel column chromatography (2-10% MeOH in CH₂Cl₂) to yield Compound 152 (0.35 g, 55%). LCMS and ¹H NMR were consistent with the desired product.

Compound 152 (0.35 g, 0.182 mmol) was dissolved in 1:1 MeOH/EtOAc (10 mL). The reaction mixture was purged by bubbling a stream of argon thru the solution for 15 minutes. Pearlman's catalyst (palladium hydroxide on carbon) was added (35 mg). Hydrogen gas was bubbled thru the solution for 30 minutes. Upon completion (TLC 10% MeOH in DCM, and LCMS), the catalyst was removed by filtration (syringe-tip Teflon filter, 0.45 μm). The filtrate was concentrated by rotary evaporation, and was dried briefly under high vacuum to yield Compound 153 (0.33 g, quantitative). The LCMS was consistent with desired product.

Compound 153 (0.33 g, 0.18 mmol) was dissolved in anhydrous DMF (5 mL) with stirring under nitrogen. To this N,N-Diisopropylethylamine (65 μL, 0.37 mmol) and PFP-TFA (35 μL, 0.28 mmol) were added. The reaction mixture was stirred under nitrogen for ˜30 min. The reaction mixture turned magenta upon contact, and gradually turned orange. The pH of the reaction mixture was maintained at pH=9-10 by adding more N,-Diisopropylethylamine. The progress of the reaction was monitored by TLC and LCMS. Upon completion, the majority of the solvent was removed under reduced pressure. The residue was diluted with CH₂Cl₂ (50 mL), and washed with saturated aqueous NaHCO₃, followed by brine. The organic layer was dried over MgSO₄, filtered, and concentrated to an orange syrup. The residue was purified by column chromatography and eluted with 2-10% MeOH in CH₂Cl₂ to yield Compound 154 (0.29 g, 79%). LCMS and ¹H NMR were consistent with the desired product.

Oligomeric Compound 155, comprising a GalNAc₃-6 conjugate group, was prepared using the general procedures illustrated in Example 46. The GalNAc₃ cluster portion of the conjugate group GalNAc₃-6 (GalNAc₃-6_(a)) can be combined with any cleavable moiety to provide a variety of conjugate groups. In certain embodiments, the cleavable moiety is —P(═O)(OH)-A_(d)-P(═O)(OH)—.

The structure of GalNAc₃-6 (GalNAc₃-6_(a)-CM-) is shown below:

Example 52: Preparation of Oligonucleotide 160 Comprising GalNAc₃-9

Compound 156 was synthesized following the procedure described in the literature (J. Med. Chem. 2004, 47, 5798-5808).

Compound 156, (18.60 g, 29.28 mmol) was dissolved in methanol (200 mL). Palladium on carbon (6.15 g, 10 wt %, loading (dry basis), matrix carbon powder, wet) was added. The reaction mixture was stirred at room temperature under hydrogen for 18 h. The reaction mixture was filtered through a pad of celite and the celite pad was washed thoroughly with methanol. The combined filtrate was washed and concentrated to dryness. The residue was purified by silica gel column chromatography and eluted with 5-10% methanol in dichloromethane to yield Compound 157 (14.26 g, 89%). Mass m/z 544.1 [M−H]⁻.

Compound 157 (5 g, 9.17 mmol) was dissolved in anhydrous DMF (30 mL). HBTU (3.65 g, 9.61 mmol) and N,N-Diisopropylethylamine (13.73 mL, 78.81 mmol) were added and the reaction mixture was stirred at room temperature for 5 minutes. To this a solution of compound 47 (2.96 g, 7.04 mmol) was added. The reaction was stirred at room temperature for 8 h. The reaction mixture was poured into a saturated NaHCO₃ aqueous solution. The mixture was extracted with ethyl acetate and the organic layer was washed with brine and dried (Na₂SO₄), filtered and evaporated. The residue obtained was purified by silica gel column chromatography and eluted with 50% ethyl acetate in hexane to yield compound 158 (8.25 g, 73.3%). The structure was confirmed by MS and ¹H NMR analysis.

Compound 158 (7.2 g, 7.61 mmol) was dried over P₂O₅ under reduced pressure. The dried compound was dissolved in anhydrous DMF (50 mL). To this 1H-tetrazole (0.43 g, 6.09 mmol) and N-methylimidazole (0.3 mL, 3.81 mmol) and 2-cyanoethyl-N,N,N′,N′-tetraisopropyl phosphorodiamidite (3.65 mL, 11.50 mmol) were added. The reaction mixture was stirred t under an argon atmosphere for 4 h. The reaction mixture was diluted with ethyl acetate (200 mL). The reaction mixture was washed with saturated NaHCO₃ and brine. The organic phase was separated, dried (Na₂SO₄), filtered and evaporated. The residue was purified by silica gel column chromatography and eluted with 50-90% ethyl acetate in hexane to yield Compound 159 (7.82 g, 80.5%). The structure was confirmed by LCMS and ³¹P NMR analysis.

Oligomeric Compound 160, comprising a GalNAc₃-9 conjugate group, was prepared using standard oligonucleotide synthesis procedures. Three units of compound 159 were coupled to the solid support, followed by nucleotide phosphoramidites. Treatment of the protected oligomeric compound with aqueous ammonia yielded compound 160. The GalNAc₃ cluster portion of the conjugate group GalNAc₃-9 (GalNAc₃-9_(a)) can be combined with any cleavable moiety to provide a variety of conjugate groups. In certain embodiments, the cleavable moiety is —P(═O)(OH)-A_(d)-P(═O)(OH)—. The structure of GalNAc₃-9 (GalNAc₃-9_(a)-CM) is shown below:

Example 53: Alternate Procedure for Preparation of Compound 18 (GalNAc₃-1a and GalNAc₃-3a)

Lactone 161 was reacted with diamino propane (3-5 eq) or Mono-Boc protected diamino propane (1 eq) to provide alcohol 162a or 162b. When unprotected propanediamine was used for the above reaction, the excess diamine was removed by evaporation under high vacuum and the free amino group in 162a was protected using CbzC1 to provide 162b as a white solid after purification by column chromatography. Alcohol 162b was further reacted with compound 4 in the presence of TMSOTfto provide 163a which was converted to 163b by removal of the Cbz group using catalytic hydrogenation. The pentafluorophenyl (PFP) ester 164 was prepared by reacting triacid 113 (see Example 48) with PFPTFA (3.5 eq) and pyridine (3.5 eq) in DMF (0.1 to 0.5 M). The triester 164 was directly reacted with the amine 163b (3-4 eq) and DIPEA (3-4 eq) to provide Compound 18. The above method greatly facilitates purification of intermediates and minimizes the formation of byproducts which are formed using the procedure described in Example 4.

Example 54: Alternate Procedure for Preparation of Compound 18 (GalNAc₃-1a and GalNAc₃-3a)

The triPFP ester 164 was prepared from acid 113 using the procedure outlined in example 53 above and reacted with mono-Boc protected diamine to provide 165 in essentially quantitative yield. The Boc groups were removed with hydrochloric acid or trifluoroacetic acid to provide the triamine which was reacted with the PFP activated acid 166 in the presence of a suitable base such as DIPEA to provide Compound 18.

The PFP protected Gal-NAc acid 166 was prepared from the corresponding acid by treatment with PFPTFA (1-1.2 eq) and pyridine (1-1.2 eq) in DMF. The precursor acid in turn was prepared from the corresponding alcohol by oxidation using TEMPO (0.2 eq) and BAIB in acetonitrile and water. The precursor alcohol was prepared from sugar intermediate 4 by reaction with 1,6-hexanediol (or 1,5-pentanediol or other diol for other n values) (2-4 eq) and TMSOTf using conditions described previously in example 47.

Example 55: Dose-Dependent Study of Oligonucleotides Comprising Either a 3′ or 5′-Conjugate Group (Comparison of GalNAc₃-1, 3, 8 and 9) Targeting SRB-1 In Vivo

The oligonucleotides listed below were tested in a dose-dependent study for antisense inhibition of SRB-1 in mice. Unconjugated ISIS 353382 was included as a standard. Each of the various GalNAc₃ conjugate groups was attached at either the 3′ or 5′ terminus of the respective oligonucleotide by a phosphodiester linked 2′-deoxyadenosine nucleoside (cleavable moiety).

TABLE 39 Modified ASO targeting SRB-1 SEQ ID ASO Sequence (5′ to 3′) Motif Conjugate No. ISIS 353382 G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) 5/10/5 none 4886 (parent) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) ISIS 655861 G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) 5/10/5 GalNAc ₃ -1 4887 ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(eo) A _(do′) -GalNAc ₃ -1 _(a) ISIS 664078 G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) 5/10/5 GalNAc ₃ -9 4887 ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(eo) A _(do′) -GalNAc ₃ -9 _(a) ISIS 661161 GalNAc ₃ -3 _(a) - _(o′) A _(do) 5/10/5 GalNAc ₃ -3 4888 G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) ISIS 665001 GalNAc ₃ -8 _(a) - _(o′) A _(do) 5/10/5 GalNAc ₃ -8 4888 G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) Capital letters indicate the nucleobase for each nucleoside and ^(m)C indicates a 5-methyl cytosine. Subscripts: “e” indicates a 2′-MOE modified nucleoside; “d” indicates a β-D-2′-deoxyribonucleoside; “s” indicates a phosphorothioate internucleoside linkage (PS); “o” indicates a phosphodiester internucleoside linkage (PO); and “o′” indicates —O—P(═O)(OH)—. Conjugate groups are in bold.

The structure of GalNAc₃-1a was shown previously in Example 9. The structure of GalNAc₃-9 was shown previously in Example 52. The structure of GalNAc₃-3 was shown previously in Example 39. The structure of GalNAc₃-8 was shown previously in Example 47.

Treatment

Six week old male Balb/c mice (Jackson Laboratory, Bar Harbor, Me.) were injected subcutaneously once at the dosage shown below with ISIS 353382, 655861, 664078, 661161, 665001 or with saline. Each treatment group consisted of 4 animals. The mice were sacrificed 72 hours following the final administration to determine the liver SRB-1 mRNA levels using real-time PCR and RIBOGREEN® RNA quantification reagent (Molecular Probes, Inc. Eugene, Oreg.) according to standard protocols. The results below are presented as the average percent of SRB-1 mRNA levels for each treatment group, normalized to the saline control.

As illustrated in Table 40, treatment with antisense oligonucleotides lowered SRB-1 mRNA levels in a dose-dependent manner. Indeed, the antisense oligonucleotides comprising the phosphodiester linked GalNAc₃-1 and GalNAc₃-9 conjugates at the 3′ terminus (ISIS 655861 and ISIS 664078) and the GalNAc₃-3 and GalNAc₃-8 conjugates linked at the 5′ terminus (ISIS 661161 and ISIS 665001) showed substantial improvement in potency compared to the unconjugated antisense oligonucleotide (ISIS 353382). Furthermore, ISIS 664078, comprising a GalNAc₃-9 conjugate at the 3′ terminus was essentially equipotent compared to ISIS 655861, which comprises a GalNAc₃-1 conjugate at the 3′ terminus. The 5′ conjugated antisense oligonucleotides, ISIS 661161 and ISIS 665001, comprising a GalNAc₃-3 or GalNAc₃-9, respectively, had increased potency compared to the 3′ conjugated antisense oligonucleotides (ISIS 655861 and ISIS 664078).

TABLE 40 ASOs containing GalNAc₃-1, 3, 8 or 9 targeting SRB-1 Dosage SRB-1 mRNA ISIS No. (mg/kg) (% Saline) Conjugate Saline n/a 100 353382 3 88 none 10 68 30 36 655861 0.5 98 GalNac₃-1 (3′) 1.5 76 5 31 15 20 664078 0.5 88 GalNac₃-9 (3′) 1.5 85 5 46 15 20 661161 0.5 92 GalNac₃-3 (5′) 1.5 59 5 19 15 11 665001 0.5 100 GalNac₃-8 (5′) 1.5 73 5 29 15 13

Liver transaminase levels, alanine aminotransferase (ALT) and aspartate aminotransferase (AST), in serum were measured relative to saline injected mice using standard protocols. Total bilirubin and BUN were also evaluated. The change in body weights was evaluated with no significant change from the saline group. ALTs, ASTs, total bilirubin and BUN values are shown in the table below.

TABLE 41 Dosage Total ISIS No. mg/kg ALT AST Bilirubin BUN Conjugate Saline 24 59 0.1 37.52 353382 3 21 66 0.2 34.65 none 10 22 54 0.2 34.2 30 22 49 0.2 33.72 655861 0.5 25 62 0.2 30.65 GalNac₃-1 (3′) 1.5 23 48 0.2 30.97 5 28 49 0.1 32.92 15 40 97 0.1 31.62 664078 0.5 40 74 0.1 35.3 GalNac₃-9 (3′) 1.5 47 104 0.1 32.75 5 20 43 0.1 30.62 15 38 92 0.1 26.2 661161 0.5 101 162 0.1 34.17 GalNac₃-3 (5′) 1.5 g 42 100 0.1 33.37   5 g 23 99 0.1 34.97 15 53 83 0.1 34.8 665001 0.5 28 54 0.1 31.32 GalNac₃-8 (5′) 1.5 42 75 0.1 32.32 5 24 42 0.1 31.85 15 32 67 0.1 31.

Example 56: Dose-Dependent Study of Oligonucleotides Comprising Either a 3′ or 5′-Conjugate Group (Comparison of GalNAc₃-1, 2, 3, 5, 6, 7 and 10) Targeting SRB-1 In Vivo

The oligonucleotides listed below were tested in a dose-dependent study for antisense inhibition of SRB-1 in mice. Unconjugated ISIS 353382 was included as a standard. Each of the various GalNAc₃ conjugate groups was attached at the 5′ terminus of the respective oligonucleotide by a phosphodiester linked 2′-deoxyadenosine nucleoside (cleavable moiety) except for ISIS 655861 which had the GalNAc₃ conjugate group attached at the 3′ terminus.

TABLE 42 Modified ASO targeting SRB-1 SEQ ASO Sequence (5′ to 3′) Motif Conjugate ID No. ISIS 353382 G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) 5/10/5 no conjugate 4886 (parent) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) ISIS 655861 G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) 5/10/5 GalNAc ₃ -1 4887 ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(eo) A _(do)′-GalNAc ₃ -1a ISIS 664507 GalNAc ₃ -2 _(a) - _(o)′A _(do)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) 5/10/5 GalNAc ₃ -2 4888 ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) ISIS 661161 GalNAc ₃ -3 _(a) - _(o)′A _(do) 5/10/5 GalNAc ₃ -3 4888 G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) ISIS 666224 GalNAc ₃ -5 _(a) - _(o)′A _(do)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) 5/10/5 GalNAc ₃ -5 4888 ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) ISIS 666961 GalNAc ₃ -6 _(a) - _(o)′A _(do)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) 5/10/5 GalNAc ₃ -6 4888 ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) ISIS 666981 GalNAc ₃ -7 _(a) - _(o)′A _(do)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) 5/10/5 GalNAc ₃ -7 4888 ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) ISIS 666881 GalNAc ₃ -10 _(a) - _(o)′A _(do)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) 5/10/5 GalNAc ₃ -10 4888 ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) Capital letters indicate the nucleobase for each nucleoside and ^(m)C indicates a 5-methyl cytosine. Subscripts: “e” indicates a 2′-MOE modified nucleoside; “d” indicates a β-D-2′-deoxyribonucleoside; “s” indicates a phosphorothioate internucleoside linkage (PS); “o” indicates a phosphodiester internucleoside linkage (PO); and “o′” indicates —O—P(═O)(OH)—. Conjugate groups are in bold.

The structure of GalNAc₃-1_(a) was shown previously in Example 9. The structure of GalNAc₃-2_(a) was shown previously in Example 37. The structure of GalNAc₃-3_(a) was shown previously in Example 39. The structure of GalNAc₃-5_(a) was shown previously in Example 49. The structure of GalNAc₃-6_(a) was shown previously in Example 51. The structure of GalNAc₃-7_(a) was shown previously in Example 48. The structure of GalNAc₃-10_(a) was shown previously in Example 46.

Treatment

Six week old male Balb/c mice (Jackson Laboratory, Bar Harbor, Me.) were injected subcutaneously once at the dosage shown below with ISIS 353382, 655861, 664507, 661161, 666224, 666961, 666981, 666881 or with saline. Each treatment group consisted of 4 animals. The mice were sacrificed 72 hours following the final administration to determine the liver SRB-1 mRNA levels using real-time PCR and RIBOGREEN RNA quantification reagent (Molecular Probes, Inc. Eugene, Oreg.) according to standard protocols. The results below are presented as the average percent of SRB-1 mRNA levels for each treatment group, normalized to the saline control.

As illustrated in Table 43, treatment with antisense oligonucleotides lowered SRB-1 mRNA levels in a dose-dependent manner. Indeed, the conjugated antisense oligonucleotides showed substantial improvement in potency compared to the unconjugated antisense oligonucleotide (ISIS 353382). The 5′ conjugated antisense oligonucleotides showed a slight increase in potency compared to the 3′ conjugated antisense oligonucleotide.

TABLE 43 Dosage SRB-1 mRNA ISIS No. (mg/kg) (% Saline) Conjugate Saline n/a 100.0 353382 3 96.0 none 10 73.1 30 36.1 655861 0.5 99.4 GalNac₃-1 (3′) 1.5 81.2 5 33.9 15 15.2 664507 0.5 102.0 GalNac₃-2 (5′) 1.5 73.2 5 31.3 15 10.8 661161 0.5 90.7 GalNac₃-3 (5′) 1.5 67.6 5 24.3 15 11.5 666224 0.5 96.1 GalNac₃-5 (5′) 1.5 61.6 5 25.6 15 11.7 666961 0.5 85.5 GalNAc₃-6 (5′) 1.5 56.3 5 34.2 15 13.1 666981 0.5 84.7 GalNAc₃-7 (5′) 1.5 59.9 5 24.9 15 8.5 666881 0.5 100.0 GalNAc₃-10 (5′) 1.5 65.8 5 26.0 15 13.0

Liver transaminase levels, alanine aminotransferase (ALT) and aspartate aminotransferase (AST), in serum were measured relative to saline injected mice using standard protocols. Total bilirubin and BUN were also evaluated. The change in body weights was evaluated with no significant change from the saline group. ALTs, ASTs, total bilirubin and BUN values are shown in Table 44 below.

TABLE 44 Dosage Total ISIS No. mg/kg ALT AST Bilirubin BUN Conjugate Saline 26 57 0.2 27 353382 3 25 92 0.2 27 none 10 23 40 0.2 25 30 29 54 0.1 28 655861 0.5 25 71 0.2 34 GalNac₃-1 (3′) 1.5 28 60 0.2 26 5 26 63 0.2 28 15 25 61 0.2 28 664507 0.5 25 62 0.2 25 GalNac₃-2 (5′) 1.5 24 49 0.2 26 5 21 50 0.2 26 15 59 84 0.1 22 661161 0.5 20 42 0.2 29 GalNac₃-3 (5′) 1.5 g 37 74 0.2 25   5 g 28 61 0.2 29 15 21 41 0.2 25 666224 0.5 34 48 0.2 21 GalNac₃-5 (5′) 1.5 23 46 0.2 26 5 24 47 0.2 23 15 32 49 0.1 26 666961 0.5 17 63 0.2 26 GalNAc₃-6 (5′) 1.5 23 68 0.2 26 5 25 66 0.2 26 15 29 107 0.2 28 666981 0.5 24 48 0.2 26 GalNAc₃-7 (5′) 1.5 30 55 0.2 24 5 46 74 0.1 24 15 29 58 0.1 26 666881 0.5 20 65 0.2 27 GalNAc₃-10 (5′) 1.5 23 59 0.2 24 5 45 70 0.2 26 15 21 57 0.2 24

Example 57: Duration of Action Study of Oligonucleotides Comprising a 3′-Conjugate Group Targeting ApoC III In Vivo

Mice were injected once with the doses indicated below and monitored over the course of 42 days for ApoC-III and plasma triglycerides (Plasma TG) levels. The study was performed using 3 transgenic mice that express human APOC-III in each group.

TABLE 45 Modified ASO targeting ApoC III SEQ ID ASO Sequence (5′ to 3′) Linkages No. ISIS A_(es)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(ds)T_(ds)T_(ds)G_(ds)T_(ds) PS 4878 304801 ^(m)C_(ds) ^(m)C_(ds)A_(ds)G_(ds) ^(m)C_(ds)T_(es)T_(es)T_(es)A_(es)T_(e) ISIS A_(es)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(ds)T_(ds)T_(ds)G_(ds)T_(ds) ^(m)C_(ds) ^(m)C_(ds) PS 4879 647535 A_(ds)G_(ds) ^(m)C_(ds)T_(es)T_(es)T_(es)A_(es)T_(eo) A _(do)′-GalNAc ₃ -1 _(a) ISIS A_(es)G_(eo) ^(m)C_(eo)T_(eo)T_(eo) ^(m)C_(ds)T_(ds)T_(ds)G_(ds)T_(ds) ^(m)C_(ds) ^(m)C_(ds) PO/PS 4879 647536 A_(ds)G_(ds) ^(m)C_(ds)T_(eo)T_(eo)T_(es)A_(es)T_(eo) A _(do)′-GalNAc ₃ -1 _(a) Capital letters indicate the nucleobase for each nucleoside and ^(m)C indicates a 5-methyl cytosine. Subscripts: “e” indicates a 2′-MOE modified nucleoside; “d” indicates a β-D-2′-deoxyribonucleoside; “s” indicates a phosphorothioate internucleoside linkage (PS); “o” indicates a phosphodiester internucleoside linkage (PO); and “o′” indicates —O—P(═O)(OH)—. Conjugate groups are in bold.

The structure of GalNAc₃-1_(a) was shown previously in Example 9.

TABLE 46 ApoC III mRNA (% Saline on Day 1) and Plasma TG Levels (% Saline on Day 1) ASO Dose Target Day 3 Day 7 Day 14 Day 35 Day 42 Saline  0 mg/kg ApoC-III 98 100 100 95 116 ISIS 30 mg/kg ApoC-III 28 30 41 65 74 304801 ISIS 10 mg/kg ApoC-III 16 19 25 74 94 647535 ISIS 10 mg/kg ApoC-III 18 16 17 35 51 647536 Saline  0 mg/kg Plasma TG 121 130 123 105 109 ISIS 30 mg/kg Plasma TG 34 37 50 69 69 304801 ISIS 10 mg/kg Plasma TG 18 14 24 18 71 647535 ISIS 10 mg/kg Plasma TG 21 19 15 32 35 647536

As can be seen in the table above the duration of action increased with addition of the 3′-conjugate group compared to the unconjugated oligonucleotide. There was a further increase in the duration of action for the conjugated mixed PO/PS oligonucleotide 647536 as compared to the conjugated full PS oligonucleotide 647535.

Example 58: Dose-Dependent Study of Oligonucleotides Comprising a 3′-Conjugate Group (Comparison of GalNAc₃-1 and GalNAc₄-11) Targeting SRB-1 In Vivo

The oligonucleotides listed below were tested in a dose-dependent study for antisense inhibition of SRB-1 in mice. Unconjugated ISIS 440762 was included as an unconjugated standard. Each of the conjugate groups were attached at the 3′ terminus of the respective oligonucleotide by a phosphodiester linked 2′-deoxyadenosine nucleoside cleavable moiety.

The structure of GalNAc₃-1_(a) was shown previously in Example 9. The structure of GalNAc₃-11_(a) was shown previously in Example 50.

Treatment

Six week old male Balb/c mice (Jackson Laboratory, Bar Harbor, Me.) were injected subcutaneously once at the dosage shown below with ISIS 440762, 651900, 663748 or with saline. Each treatment group consisted of 4 animals. The mice were sacrificed 72 hours following the final administration to determine the liver SRB-1 mRNA levels using real-time PCR and RIBOGREEN® RNA quantification reagent (Molecular Probes, Inc. Eugene, Oreg.) according to standard protocols. The results below are presented as the average percent of SRB-1 mRNA levels for each treatment group, normalized to the saline control.

As illustrated in Table 47, treatment with antisense oligonucleotides lowered SRB-1 mRNA levels in a dose-dependent manner. The antisense oligonucleotides comprising the phosphodiester linked GalNAc₃-1 and GalNAc₄-11 conjugates at the 3′ terminus (ISIS 651900 and ISIS 663748) showed substantial improvement in potency compared to the unconjugated antisense oligonucleotide (ISIS 440762). The two conjugated oligonucleotides, GalNAc₃-1 and GalNAc₄-11, were equipotent.

TABLE 47 Modified ASO targeting SRB-1 % Saline SEQ ID ASO Sequence (5′ to 3′) Dose mg/kg control No. Saline 100 ISIS 440762 T_(ks) ^(m)C_(ks)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) 0.6 73.45 4880 ^(m)C_(ds)T_(ds)T_(ks) ^(m)C_(k) 2 59.66 6 23.50 ISIS 651900 T_(ks) ^(m)C_(ks)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) 0.2 62.75 4881 ^(m)C_(ds)T_(ds)T_(ks) ^(m)C_(ko) A _(do)′-GalNAc ₃ -1 _(a) 0.6 29.14 2 8.61 6 5.62 ISIS 663748 T_(ks) ^(m)C_(ks)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) 0.2 63.99 4881 ^(m)C_(ds)T_(ds)T_(ks) ^(m)C_(ko) A _(do)′-GalNAc ₄ -11 _(a) 0.6 33.53 2 7.58 6 5.52 Capital letters indicate the nucleobase for each nucleoside and ^(m)C indicates a 5-methyl cytosine. Subscripts: “e” indicates a 2′-MOE modified nucleoside; “k” indicates 6′-(S)—CH₃ bicyclic nucleoside; “d” indicates a β-D-2′-deoxyribonucleoside; “s” indicates a phosphorothioate internucleoside linkage (PS); “o” indicates a phosphodiester internucleoside linkage (PO); and “o′” indicates —O—P(═O)(OH)—. Conjugate groups are in bold.

Liver transaminase levels, alanine aminotransferase (ALT) and aspartate aminotransferase (AST), in serum were measured relative to saline injected mice using standard protocols. Total bilirubin and BUN were also evaluated. The change in body weights was evaluated with no significant change from the saline group. ALTs, ASTs, total bilirubin and BUN values are shown in Table 48 below.

TABLE 48 Dosage Total ISIS No. mg/kg ALT AST Bilirubin BUN Conjugate Saline 30 76 0.2 40 440762 0.60 32 70 0.1 35 none 2 26 57 0.1 35 6 31 48 0.1 39 651900 0.2 32 115 0.2 39 GalNac₃-1 (3′) 0.6 33 61 0.1 35 2 30 50 0.1 37 6 34 52 0.1 36 663748 0.2 28 56 0.2 36 GalNac₄-11 (3′) 0.6 34 60 0.1 35 2 44 62 0.1 36 6 38 71 0.1 33

Example 59: Effects of GalNAc₃-1 Conjugated ASOs Targeting FXI In Vivo

The oligonucleotides listed below were tested in a multiple dose study for antisense inhibition of FXI in mice. ISIS 404071 was included as an unconjugated standard. Each of the conjugate groups was attached at the 3′ terminus of the respective oligonucleotide by a phosphodiester linked 2′-deoxyadenosine nucleoside cleavable moiety.

TABLE 49 Modified ASOs targeting FXI SEQ ID ASO Sequence (5′ to 3′) Linkages No. ISIS T_(es)G_(es)G_(es)T_(es)A_(es)A_(ds)T_(ds) ^(m)C_(ds) ^(m)C_(ds)A_(ds) ^(m)C_(ds) PS 4889 404071 T_(ds)T_(ds)T_(ds) ^(m)C_(ds)A_(es)G_(es)A_(es)G_(es)G_(e) ISIS T_(es)G_(es)G_(es)T_(es)A_(es)A_(ds)T_(ds) ^(m)C_(ds) ^(m)C_(ds)A_(ds) ^(m)C_(ds) PS 4890 656172 T_(ds)T_(ds)T_(ds) ^(m)C_(ds)A_(es)G_(es)A_(es)G_(es)G_(eo) A _(do)′-GalNAc ₃ -1 _(a) ISIS T_(es)G_(eo)G_(eo)T_(eo)A_(eo)A_(ds)T_(ds) ^(m)C_(ds) ^(m)C_(ds)A_(ds) ^(m)C_(ds) PO/PS 4890 656173 T_(ds)T_(ds)T_(ds) ^(m)C_(ds)A_(eo)G_(eo)A_(es)G_(es)G_(eo) A _(do)′-GalNAc ₃ -1 _(a) Capital letters indicate the nucleobase for each nucleoside and ^(m)C indicates a 5-methyl cytosine. Subscripts: “e” indicates a 2′-MOE modified nucleoside; “d” indicates a β-D-2′-deoxyribonucleoside; “s” indicates a phosphorothioate internucleoside linkage (PS); “o” indicates a phosphodiester internucleoside linkage (PO); and “o′” indicates —O—P(═O)(OH)—. Conjugate groups are in bold.

The structure of GalNAc₃-1_(a) was shown previously in Example 9.

Treatment

Six week old male Balb/c mice (Jackson Laboratory, Bar Harbor, Me.) were injected subcutaneously twice a week for 3 weeks at the dosage shown below with ISIS 404071, 656172, 656173 or with PBS treated control. Each treatment group consisted of 4 animals. The mice were sacrificed 72 hours following the final administration to determine the liver FXI mRNA levels using real-time PCR and RIBOGREEN RNA quantification reagent (Molecular Probes, Inc. Eugene, Oreg.) according to standard protocols. Plasma FXI protein levels were also measured using ELISA. FXI mRNA levels were determined relative to total RNA (using RIBOGREEN®), prior to normalization to PBS-treated control. The results below are presented as the average percent of FXI mRNA levels for each treatment group. The data was normalized to PBS-treated control and is denoted as “% PBS”. The ED₅₀s were measured using similar methods as described previously and are presented below.

TABLE 50 Factor XI mRNA (% Saline) Dose ASO mg/kg % Control Conjugate Linkages Saline 100 none ISIS 3 92 none PS 404071 10 40 30 15 ISIS 0.7 74 GalNAc₃-1 PS 656172 2 33 6 9 ISIS 0.7 49 GalNAc₃-1 PO/PS 656173 2 22 6 1

As illustrated in Table 50, treatment with antisense oligonucleotides lowered FXI mRNA levels in a dose-dependent manner. The oligonucleotides comprising a 3′-GalNAc₃-1 conjugate group showed substantial improvement in potency compared to the unconjugated antisense oligonucleotide (ISIS 404071). Between the two conjugated oligonucleotides an improvement in potency was further provided by substituting some of the PS linkages with PO (ISIS 656173).

As illustrated in Table 50a, treatment with antisense oligonucleotides lowered FXI protein levels in a dose-dependent manner. The oligonucleotides comprising a 3′-GalNAc₃-1 conjugate group showed substantial improvement in potency compared to the unconjugated antisense oligonucleotide (ISIS 404071). Between the two conjugated oligonucleotides an improvement in potency was further provided by substituting some of the PS linkages with PO (ISIS 656173).

TABLE 50a Factor XI protein (% Saline) Dose Protein ASO mg/kg (% Control) Conjugate Linkages Saline 100 none ISIS 3 127 none PS 404071 10 32 30 3 ISIS 0.7 70 GalNAc₃-1 PS 656172 2 23 6 1 ISIS 0.7 45 GalNAc₃-1 PO/PS 656173 2 6 6 0

Liver transaminase levels, alanine aminotransferase (ALT) and aspartate aminotransferase (AST), in serum were measured relative to saline injected mice using standard protocols. Total bilirubin, total albumin, CRE and BUN were also evaluated. The change in body weights was evaluated with no significant change from the saline group. ALTs, ASTs, total bilirubin and BUN values are shown in the table below.

TABLE 51 Dosage Total Total ISIS No. mg/kg ALT AST Albumin Bilirubin CRE BUN Conjugate Saline 71.8 84.0 3.1 0.2 0.2 22.9 404071 3 152.8 176.0 3.1 0.3 0.2 23.0 none 10 73.3 121.5 3.0 0.2 0.2 21.4 30 82.5 92.3 3.0 0.2 0.2 23.0 656172 0.7 62.5 111.5 3.1 0.2 0.2 23.8 GalNac₃-1 (3′) 2 33.0 51.8 2.9 0.2 0.2 22.0 6 65.0 71.5 3.2 0.2 0.2 23.9 656173 0.7 54.8 90.5 3.0 0.2 0.2 24.9 GalNac₃-1 (3′) 2 85.8 71.5 3.2 0.2 0.2 21.0 6 114.0 101.8 3.3 0.2 0.2 22.7

Example 60: Effects of Conjugated ASOs Targeting SRB-1 In Vitro

The oligonucleotides listed below were tested in a multiple dose study for antisense inhibition of SRB-1 in primary mouse hepatocytes. ISIS 353382 was included as an unconjugated standard. Each of the conjugate groups were attached at the 3′ or 5′ terminus of the respective oligonucleotide by a phosphodiester linked 2′-deoxyadenosine nucleoside cleavable moiety.

TABLE 52 Modified ASO targeting SRB-1 SEQ ASO Sequence (5′ to 3′) Motif Conjugate ID No. ISIS 353382 G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) 5/10/5 none 4886 ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) ISIS 655861 G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) 5/10/5 GalNAc ₃ -1 4887 ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(eo) A _(do)′-GalNAc ₃ -1 _(a) ISIS 655862 G_(es) ^(m)C_(eo)T_(eo)T_(eo) ^(m)C_(eo)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) 5/10/5 GalNAc ₃ -1 4887 ^(m)C_(ds)T_(ds)T_(eo) ^(m)C_(eo) ^(m)C_(es)T_(es)T_(eo) A _(do)′-GalNAc ₃ -1 _(a) ISIS 661161 GalNAc ₃ -3 _(a-o)′A _(do)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds) 5/10/5 GalNAc ₃ -3 4888 T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) ISIS 665001 GalNAc ₃ -8 _(a-o)′A _(do)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds) 5/10/5 GalNAc ₃ -8 4888 T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) ISIS 664078 G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) 5/10/5 GalNAc ₃ -9 4887 ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(eo) A _(do)′-GalNAc ₃ -9 _(a) ISIS 666961 GalNAC ₃ -6 _(a) - _(o)′A _(do)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds) 5/10/5 GalNAc ₃ -6 4888 T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) ISIS 664507 GalNAc ₃ -2 _(a) - _(o)′A _(do)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) 5/10/5 GalNAc ₃ -2 4888 ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) ISIS 666881 GalNAc ₃ -10 _(a) - _(o)′A _(do)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) 5/10/5 GalNAc ₃ -10 4888 ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) ISIS 666224 GalNAc ₃ -5 _(a) - _(o)′A _(do)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) 5/10/5 GalNAc ₃ -5 4888 ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) ISIS 666981 GalNAc ₃ -7 _(a) - _(o)′A _(do)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) 5/10/5 GalNAc ₃ -7 4888 ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) Capital letters indicate the nucleobase for each nucleoside and ^(m)C indicates a 5-methyl cytosine. Subscripts: “e” indicates a 2′-MOE modified nucleoside; “d” indicates a β-D-2′-deoxyribonucleoside; “s” indicates a phosphorothioate internucleoside linkage (PS); “o” indicates a phosphodiester internucleoside linkage (PO); and “o′” indicates —O—P(═O)(OH)—. Conjugate groups are in bold.

The structure of GalNAc₃-1_(a) was shown previously in Example 9. The structure of GalNAc₃-3a was shown previously in Example 39. The structure of GalNAc₃-8a was shown previously in Example 47. The structure of GalNAc₃-9a was shown previously in Example 52. The structure of GalNAc₃-6a was shown previously in Example 51. The structure of GalNAc₃-2a was shown previously in Example 37. The structure of GalNAc₃-10a was shown previously in Example 46. The structure of GalNAc₃-5a was shown previously in Example 49. The structure of GalNAc₃-7a was shown previously in Example 48.

Treatment

The oligonucleotides listed above were tested in vitro in primary mouse hepatocyte cells plated at a density of 25,000 cells per well and treated with 0.03, 0.08, 0.24, 0.74, 2.22, 6.67 or 20 nM modified oligonucleotide. After a treatment period of approximately 16 hours, RNA was isolated from the cells and mRNA levels were measured by quantitative real-time PCR and the SRB-1 mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®.

The IC₅₀ was calculated using standard methods and the results are presented in Table 53. The results show that, under free uptake conditions in which no reagents or electroporation techniques are used to artificially promote entry of the oligonucleotides into cells, the oligonucleotides comprising a GalNAc conjugate were significantly more potent in hepatocytes than the parent oligonucleotide (ISIS 353382) that does not comprise a GalNAc conjugate.

TABLE 53 IC₅₀ Internucleoside SEQ ASO (nM) linkages Conjugate ID No. ISIS 353382   190^(a) PS none 4886 ISIS 655861  11^(a) PS GalNAc₃-1 4887 ISIS 655862  3 PO/PS GalNAc₃-1 4887 ISIS 661161  15^(a) PS GalNAc₃-3 4888 ISIS 665001 20 PS GalNAc₃-8 4888 ISIS 664078 55 PS GalNAc₃-9 4887 ISIS 666961  22^(a) PS GalNAc₃-6 4888 ISIS 664507 30 PS GalNAc₃-2 4888 ISIS 666881 30 PS GalNAc₃-10 4888 ISIS 666224  30^(a) PS GalNAc₃-5 4888 ISIS 666981 40 PS GalNAc₃-7 4888 ^(a)Average of multiple runs.

Example 61: Preparation of Oligomeric Compound 175 Comprising GalNAc₃-12

Compound 169 is commercially available. Compound 172 was prepared by addition of benzyl (perfluorophenyl) glutarate to compound 171. The benzyl (perfluorophenyl) glutarate was prepared by adding PFP-TFA and DIEA to 5-(benzyloxy)-5-oxopentanoic acid in DMF. Oligomeric compound 175, comprising a GalNAc₃-12 conjugate group, was prepared from compound 174 using the general procedures illustrated in Example 46. The GalNAc₃ cluster portion of the conjugate group GalNAc₃-12 (GalNAc₃-12_(a)) can be combined with any cleavable moiety to provide a variety of conjugate groups. In a certain embodiments, the cleavable moiety is —P(═O)(OH)-A_(d)-P(═O)(OH)—. The structure of GalNAc₃-12 (GalNAc₃-12_(a)-CM-) is shown below:

Example 62: Preparation of Oligomeric Compound 180 Comprising GalNAc₃-13

Compound 176 was prepared using the general procedure shown in Example 2. Oligomeric compound 180, comprising a GalNAc₃-13 conjugate group, was prepared from compound 177 using the general procedures illustrated in Example 49. The GalNAc₃ cluster portion of the conjugate group GalNAc₃-13 (GalNAc₃-13a) can be combined with any cleavable moiety to provide a variety of conjugate groups. In a certain embodiments, the cleavable moiety is —P(═O)(OH)-A_(d)-P(═O)(OH)—. The structure of GalNAc₃-13 (GalNAc₃-13_(a)-CM-) is shown below:

Example 63: Preparation of Oligomeric Compound 188 Comprising GalNAc₃-14

Compounds 181 and 185 are commercially available. Oligomeric compound 188, comprising a GalNAc₃-14 conjugate group, was prepared from compound 187 using the general procedures illustrated in Example 46. The GalNAc₃ cluster portion of the conjugate group GalNAc₃-14 (GalNAc₃-14_(a)) can be combined with any cleavable moiety to provide a variety of conjugate groups. In certain embodiments, the cleavable moiety is —P(O)(OH)-A_(d)-P(O)(OH)—. The structure of GalNAc₃-14 (GalNAc₃-14_(a)-CM-) is shown below:

Example 64: Preparation of Oligomeric Compound 197 Comprising GalNAc₃-15

Compound 189 is commercially available. Compound 195 was prepared using the general procedure shown in Example 31. Oligomeric compound 197, comprising a GalNAc₃-15 conjugate group, was prepared from compounds 194 and 195 using standard oligonucleotide synthesis procedures. The GalNAc₃ cluster portion of the conjugate group GalNAc₃-15 (GalNAc₃-15_(a)) can be combined with any cleavable moiety to provide a variety of conjugate groups. In certain embodiments, the cleavable moiety is —P(═O)(OH)-A_(d)-P(═O)(OH)—. The structure of GalNAc₃-15 (GalNAc₃-15_(a)-CM-) is shown below:

Example 65: Dose-Dependent Study of Oligonucleotides Comprising a 5′-Conjugate Group (Comparison of GalNAc₃-3, 12, 13, 14, and 15) Targeting SRB-1 In Vivo

The oligonucleotides listed below were tested in a dose-dependent study for antisense inhibition of SRB-1 in mice. Unconjugated ISIS 353382 was included as a standard. Each of the GalNAc₃ conjugate groups was attached at the 5′ terminus of the respective oligonucleotide by a phosphodiester linked 2′-deoxyadenosine nucleoside (cleavable moiety).

TABLE 54 Modified ASOs targeting SRB-1 SEQ ISIS ID No. Sequences (5′ to 3′) Conjugate No. 353382 G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) none 4886 661161 GalNAc ₃ -3 _(a) - _(o)′A _(do)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds) GalNAc₃-3 4888 T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) 671144 GalNAc ₃ -12 _(a) - _(o)′A _(do)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds) GalNAc₃-12 4888 T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) 670061 GalNAc ₃ -13 _(a) - _(o)′A _(do)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds) GalNAc₃-13 4888 T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) 671261 GalNAc ₃ -14 _(a) - _(o)′A _(do)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds) GalNAc₃-14 4888 T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) 671262 GalNAc₃-15_(a)-_(o)′A_(do)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds) GalNAc₃-15 4888 T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) Capital letters indicate the nucleobase for each nucleoside and ^(m)C indicates a 5-methyl cytosine. Subscripts: “e” indicates a 2′-MOE modified nucleoside; “d” indicates a β-D-2′-deoxyribonucleoside; “s” indicates a phosphorothioate internucleoside linkage (PS); “o” indicates a phosphodiester internucleoside linkage (PO); and “o′” indicates —O—P(═O)(OH)—. Conjugate groups are in bold.

The structure of GalNAc₃-3_(a) was shown previously in Example 39. The structure of GalNAc₃-12a was shown previously in Example 61. The structure of GalNAc₃-13a was shown previously in Example 62. The structure of GalNAc₃-14a was shown previously in Example 63. The structure of GalNAc₃-15a was shown previously in Example 64.

Treatment

Six to eight week old C57bl6 mice (Jackson Laboratory, Bar Harbor, Me.) were injected subcutaneously once or twice at the dosage shown below with ISIS 353382, 661161, 671144, 670061, 671261, 671262, or with saline. Mice that were dosed twice received the second dose three days after the first dose. Each treatment group consisted of 4 animals. The mice were sacrificed 72 hours following the final administration to determine the liver SRB-1 mRNA levels using real-time PCR and RIBOGREEN® RNA quantification reagent (Molecular Probes, Inc. Eugene, Oreg.) according to standard protocols. The results below are presented as the average percent of SRB-1 mRNA levels for each treatment group, normalized to the saline control.

As illustrated in Table 55, treatment with antisense oligonucleotides lowered SRB-1 mRNA levels in a dose-dependent manner. No significant differences in target knockdown were observed between animals that received a single dose and animals that received two doses (see ISIS 353382 dosages 30 and 2×15 mg/kg; and ISIS 661161 dosages 5 and 2×2.5 mg/kg). The antisense oligonucleotides comprising the phosphodiester linked GalNAc₃-3, 12, 13, 14, and 15 conjugates showed substantial improvement in potency compared to the unconjugated antisense oligonucleotide (ISIS 335382).

TABLE 55 SRB-1 mRNA (% Saline) Dosage SRB-1 mRNA ED₅₀ ISIS No. (mg/kg) (% Saline) (mg/kg) Conjugate Saline n/a 100.0 n/a n/a 353382 3 85.0 22.4 none 10 69.2 30 34.2 2 × 15  36.0 661161 0.5 87.4 2.2 GalNAc₃-3 1.5 59.0 5 25.6 2 × 2.5 27.5 15 17.4 671144 0.5 101.2 3.4 GalNAc₃-12 1.5 76.1 5 32.0 15 17.6 670061 0.5 94.8 2.1 GalNAc₃-13 1.5 57.8 5 20.7 15 13.3 671261 0.5 110.7 4.1 GalNAc₃-14 1.5 81.9 5 39.8 15 14.1 671262 0.5 109.4 9.8 GalNAc₃-15 1.5 99.5 5 69.2 15 36.1

Liver transaminase levels, alanine aminotransferase (ALT) and aspartate aminotransferase (AST), in serum were measured relative to saline injected mice using standard protocols. Total bilirubin and BUN were also evaluated. The changes in body weights were evaluated with no significant differences from the saline group (data not shown). ALTs, ASTs, total bilirubin and BUN values are shown in Table 56 below.

TABLE 56 Total Dosage ALT AST Bilirubin BUN ISIS No. (mg/kg) (U/L) (U/L) (mg/dL) (mg/dL) Conjugate Saline n/a 28 60 0.1 39 n/a 353382 3 30 77 0.2 36 none 10 25 78 0.2 36 30 28 62 0.2 35 2 × 15  22 59 0.2 33 661161 0.5 39 72 0.2 34 GalNAc₃-3 1.5 26 50 0.2 33 5 41 80 0.2 32 2 × 2.5 24 72 0.2 28 15 32 69 0.2 36 671144 0.5 25 39 0.2 34 GalNAc₃-12 1.5 26 55 0.2 28 5 48 82 0.2 34 15 23 46 0.2 32 670061 0.5 27 53 0.2 33 GalNAc₃-13 1.5 24 45 0.2 35 5 23 58 0.1 34 15 24 72 0.1 31 671261 0.5 69 99 0.1 33 GalNAc₃-14 1.5 34 62 0.1 33 5 43 73 0.1 32 15 32 53 0.2 30 671262 0.5 24 51 0.2 29 GalNAc₃-15 1.5 32 62 0.1 31 5 30 76 0.2 32 15 31 64 0.1 32

Example 66: Effect of Various Cleavable Moieties on Antisense Inhibition In Vivo by Oligonucleotides Targeting SRB-1 Comprising a 5′-GalNAc₃ Cluster

The oligonucleotides listed below were tested in a dose-dependent study for antisense inhibition of SRB-1 in mice. Each of the GalNAc₃ conjugate groups was attached at the 5′ terminus of the respective oligonucleotide by a phosphodiester linked nucleoside (cleavable moiety (CM)).

TABLE 57 Modified ASOs targeting SRB-1 ISIS GalNAc₃ SEQ No. Sequences (5′ to 3′) Cluster CM ID No. 661161 GalNAc ₃ -3 _(a) - _(o′) A _(do)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds) GalNAc₃-3a A_(d) 4888 G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) 670699 GalNAc ₃ -3 _(a) - _(o′) T _(do)G_(es) ^(m)C_(eo)T_(eo)T_(eo) ^(m)C_(eo)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds) GalNAc₃-3a T_(d) 4891 G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(eo) ^(m)C_(eo)C_(es)T_(es)T_(e) 670700 GalNAc ₃ -3 _(a) - _(o′) A _(eo)G_(es) ^(m)C_(eo)T_(eo)T_(eo) ^(m)C_(eo)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds) GalNAc₃-3a A_(e) 4888 G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(eo) ^(m)C_(eo) ^(m)C_(es)T_(es)T_(e) 670701 GalNAC ₃ -3 _(a) - _(o′) T _(eo)G_(es) ^(m)C_(eo)T_(eo)T_(eo) ^(m)C_(eo)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds) GalNAc₃-3a T_(e) 4891 G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(eo) ^(m)C_(eo) ^(m)C_(es)T_(es)T_(e) 671165 GalNAc ₃ -13 _(a) - _(o′) A _(do)G_(es) ^(m)C_(eo)T_(eo)T_(eo) ^(m)C_(eo)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds) GalNAc₃-13a A_(d) 4888 G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(eo) ^(m)C_(eo) ^(m)C_(es)T_(es)T_(e) Capital letters indicate the nucleobase for each nucleoside and ^(m)C indicates a 5-methyl cytosine. Subscripts: “e” indicates a 2′-MOE modified nucleoside; “d” indicates a β-D-2′-deoxyribonucleoside; “s” indicates a phosphorothioate internucleoside linkage (PS); “o” indicates a phosphodiester internucleoside linkage (PO); and “o′” indicates —O—P(═O)(OH)—. Conjugate groups are in bold.

The structure of GalNAc₃-3_(a) was shown previously in Example 39. The structure of GalNAc₃-13a was shown previously in Example 62.

Treatment

Six to eight week old C57bl6 mice (Jackson Laboratory, Bar Harbor, Me.) were injected subcutaneously once at the dosage shown below with ISIS 661161, 670699, 670700, 670701, 671165, or with saline. Each treatment group consisted of 4 animals. The mice were sacrificed 72 hours following the final administration to determine the liver SRB-1 mRNA levels using real-time PCR and RIBOGREEN® RNA quantification reagent (Molecular Probes, Inc. Eugene, Oreg.) according to standard protocols. The results below are presented as the average percent of SRB-1 mRNA levels for each treatment group, normalized to the saline control.

As illustrated in Table 58, treatment with antisense oligonucleotides lowered SRB-1 mRNA levels in a dose-dependent manner. The antisense oligonucleotides comprising various cleavable moieties all showed similar potencies.

TABLE 58 SRB-1 mRNA (% Saline) Dosage SRB-1 mRNA GalNAc₃ ISIS No. (mg/kg) (% Saline) Cluster CM Saline n/a 100.0 n/a n/a 661161 0.5 87.8 GalNAc₃-3a A_(d) 1.5 61.3 5 33.8 15 14.0 670699 0.5 89.4 GalNAc₃-3a T_(d) 1.5 59.4 5 31.3 15 17.1 670700 0.5 79.0 GalNAc₃-3a A_(e) 1.5 63.3 5 32.8 15 17.9 670701 0.5 79.1 GalNAc₃-3a T_(e) 1.5 59.2 5 35.8 15 17.7 671165 0.5 76.4 GalNAc₃-13a A_(d) 1.5 43.2 5 22.6 15 10.0

Liver transaminase levels, alanine aminotransferase (ALT) and aspartate aminotransferase (AST), in serum were measured relative to saline injected mice using standard protocols. Total bilirubin and BUN were also evaluated. The changes in body weights were evaluated with no significant differences from the saline group (data not shown). ALTs, ASTs, total bilirubin and BUN values are shown in Table 56 below.

TABLE 59 Total Dosage ALT AST Bilirubin BUN GalNAc₃ ISIS No. (mg/kg) (U/L) (U/L) (mg/dL) (mg/dL) Cluster CM Saline n/a 24 64 0.2 31 n/a n/a 661161 0.5 25 64 0.2 31 GalNAc₃-3a A_(d) 1.5 24 50 0.2 32 5 26 55 0.2 28 15 27 52 0.2 31 670699 0.5 42 83 0.2 31 GalNAc₃-3a T_(d) 1.5 33 58 0.2 32 5 26 70 0.2 29 15 25 67 0.2 29 670700 0.5 40 74 0.2 27 GalNAc₃-3a A_(e) 1.5 23 62 0.2 27 5 24 49 0.2 29 15 25 87 0.1 25 670701 0.5 30 77 0.2 27 GalNAc₃-3a T_(e) 1.5 22 55 0.2 30 5 81 101 0.2 25 15 31 82 0.2 24 671165 0.5 44 84 0.2 26 GalNAc₃-13a A_(d) 1.5 47 71 0.1 24 5 33 91 0.2 26 15 33 56 0.2 29

Example 67: Preparation of Oligomeric Compound 199 Comprising GalNAc₃-16

Oligomeric compound 199, comprising a GalNAc₃-16 conjugate group, is prepared using the general procedures illustrated in Examples 7 and 9. The GalNAc₃ cluster portion of the conjugate group GalNAc₃-16 (GalNAc₃-16_(a)) can be combined with any cleavable moiety to provide a variety of conjugate groups. In certain embodiments, the cleavable moiety is —P(═O)(OH)-A_(d)-P(═O)(OH)—. The structure of GalNAc₃-16 (GalNAc₃-16_(a)-CM-) is shown below:

Example 68: Preparation of Oligomeric Compound 200 Comprising GalNAc₃-17

Oligomeric compound 200, comprising a GalNAc₃-17 conjugate group, was prepared using the general procedures illustrated in Example 46. The GalNAc₃ cluster portion of the conjugate group GalNAc₃-17 (GalNAc₃-17_(a)) can be combined with any cleavable moiety to provide a variety of conjugate groups. In certain embodiments, the cleavable moiety is —P(═O)(OH)-A_(d)-P(═O)(OH)—. The structure of GalNAc₃-17 (GalNAc₃-17_(a)-CM-) is shown below:

Example 69: Preparation of Oligomeric Compound 201 Comprising GalNAc₃-18

Oligomeric compound 201, comprising a GalNAc₃-18 conjugate group, was prepared using the general procedures illustrated in Example 46. The GalNAc₃ cluster portion of the conjugate group GalNAc₃-18 (GalNAc₃-18_(a)) can be combined with any cleavable moiety to provide a variety of conjugate groups. In certain embodiments, the cleavable moiety is —P(═O)(OH)-A_(d)-P(═O)(OH)—. The structure of GalNAc₃-18 (GalNAc₃-18_(a)-CM-) is shown below:

Example 70: Preparation of Oligomeric Compound 204 Comprising GalNAc₃-19

Oligomeric compound 204, comprising a GalNAc₃-19 conjugate group, was prepared from compound 64 using the general procedures illustrated in Example 52. The GalNAc₃ cluster portion of the conjugate group GalNAc₃-19 (GalNAc₃-19_(a)) can be combined with any cleavable moiety to provide a variety of conjugate groups. In certain embodiments, the cleavable moiety is —P(═O)(OH)-A_(d)-P(═O)(OH)—. The structure of GalNAc₃-19 (GalNAc₃-19_(a)-CM-) is shown below:

Example 71: Preparation of Oligomeric Compound 210 Comprising GalNAc₃-20

Compound 205 was prepared by adding PFP-TFA and DIEA to 6-(2,2,2-trifluoroacetamido)hexanoic acid in acetonitrile, which was prepared by adding triflic anhydride to 6-aminohexanoic acid. The reaction mixture was heated to 80° C., then lowered to rt. Oligomeric compound 210, comprising a GalNAc₃-20 conjugate group, was prepared from compound 208 using the general procedures illustrated in Example 52. The GalNAc₃ cluster portion of the conjugate group GalNAc₃-20 (GalNAc₃-20_(a)) can be combined with any cleavable moiety to provide a variety of conjugate groups. In certain embodiments, the cleavable moiety is —P(═O)(OH)-A_(d)-P(═O)(OH)—. The structure of GalNAc₃-20 (GalNAc₃-20_(a)-CM-) is shown below:

Example 72: Preparation of Oligomeric Compound 215 Comprising GalNAc₃-21

Compound 211 is commercially available. Oligomeric compound 215, comprising a GalNAc₃-21 conjugate group, was prepared from compound 213 using the general procedures illustrated in Example 52. The GalNAc₃ cluster portion of the conjugate group GalNAc₃-21 (GalNAc₃-21_(a)) can be combined with any cleavable moiety to provide a variety of conjugate groups. In certain embodiments, the cleavable moiety is —P(═O)(OH)-A_(d)-P(═O)(OH)—. The structure of GalNAc₃-21 (GalNAc₃-2_(a)-CM-) is shown below:

Example 73: Preparation of Oligomeric Compound 221 Comprising GalNAc₃-22

Compound 220 was prepared from compound 219 using diisopropylammonium tetrazolide. Oligomeric compound 221, comprising a GalNAc₃-21 conjugate group, is prepared from compound 220 using the general procedure illustrated in Example 52. The GalNAc₃ cluster portion of the conjugate group GalNAc₃-22 (GalNAc₃-22_(a)) can be combined with any cleavable moiety to provide a variety of conjugate groups. In certain embodiments, the cleavable moiety is —P(═O)(OH)-A_(d)-P(═O)(OH)—. The structure of GalNAc₃-22 (GalNAc₃-22_(a)-CM-) is shown below:

Example 74: Effect of Various Cleavable Moieties on Antisense Inhibition In Vivo by Oligonucleotides Targeting SRB-1 Comprising a 5′-GalNAc₃ Conjugate

The oligonucleotides listed below were tested in a dose-dependent study for antisense inhibition of SRB-1 in mice. Each of the GalNAc₃ conjugate groups was attached at the 5′ terminus of the respective oligonucleotide.

TABLE 60 Modified ASOs targeting SRB-1 ISIS GalNAc₃ SEQ No. Sequences (5′ to 3′) Cluster CM ID No. 353382 G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) n/a n/a 4886 ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) 661161 GalNAc ₃ -3 _(a) - _(o′) A _(do)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds) GalNAc₃-3a A_(d) 4888 G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) 666904 GalNAc ₃ -3 _(a) - _(o′)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds) GalNAc₃-3a PO 4886 G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) 675441 GalNAc ₃ -17 _(a) - _(o′) A _(do)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds) GalNAc₃-17a A_(d) 4888 G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) 675442 GalNAc ₃ -18 _(a) - _(o′) A _(do)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)C_(ds)A_(ds)T_(ds) GalNAc₃-18a A_(d) 4888 G_(ds)A_(ds) ^(m)C_(ds)T_(ds) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e)

In all tables, capital letters indicate the nucleobase for each nucleoside and C indicates a 5-methyl cytosine. Subscripts: “e” indicates a 2′-MOE modified nucleoside; “d” indicates a β-D-2′-deoxyribonucleoside; “s” indicates a phosphorothioate internucleoside linkage (PS); “o” indicates a phosphodiester internucleoside linkage (PO); and “o′” indicates —O—P(═O)(OH)—. Conjugate groups are in bold.

The structure of GalNAc₃-3_(a) was shown previously in Example 39. The structure of GalNAc₃-17a was shown previously in Example 68, and the structure of GalNAc₃-18a was shown in Example 69.

Treatment

Six to eight week old C57BL/6 mice (Jackson Laboratory, Bar Harbor, Me.) were injected subcutaneously once at the dosage shown below with an oligonucleotide listed in Table 60 or with saline. Each treatment group consisted of 4 animals. The mice were sacrificed 72 hours following the final administration to determine the SRB-1 mRNA levels using real-time PCR and RIBOGREEN RNA quantification reagent (Molecular Probes, Inc. Eugene, Oreg.) according to standard protocols. The results below are presented as the average percent of SRB-1 mRNA levels for each treatment group, normalized to the saline control.

As illustrated in Table 61, treatment with antisense oligonucleotides lowered SRB-1 mRNA levels in a dose-dependent manner. The antisense oligonucleotides comprising a GalNAc conjugate showed similar potencies and were significantly more potent than the parent oligonucleotide lacking a GalNAc conjugate.

TABLE 61 SRB-1 mRNA (% Saline) Dosage SRB-1 mRNA GalNAc₃ ISIS No. (mg/kg) (% Saline) Cluster CM Saline n/a 100.0 n/a n/a 353382 3 79.38 n/a n/a 10 68.67 30 40.70 661161 0.5 79.18 GalNAc₃-3a A_(d) 1.5 75.96 5 30.53 15 12.52 666904 0.5 91.30 GalNAc₃-3a PO 1.5 57.88 5 21.22 15 16.49 675441 0.5 76.71 GalNAc₃-17a A_(d) 1.5 63.63 5 29.57 15 13.49 675442 0.5 95.03 GalNAc₃-18a A_(d) 1.5 60.06 5 31.04 15 19.40

Liver transaminase levels, alanine aminotransferase (ALT) and aspartate aminotransferase (AST), in serum were measured relative to saline injected mice using standard protocols. Total bilirubin and BUN were also evaluated. The change in body weights was evaluated with no significant change from the saline group (data not shown). ALTs, ASTs, total bilirubin and BUN values are shown in Table 62 below.

TABLE 62 Total Dosage ALT AST Bilirubin BUN GalNAc₃ ISIS No. (mg/kg) (U/L) (U/L) (mg/dL) (mg/dL) Cluster CM Saline n/a 26 59 0.16 42 n/a n/a 353382 3 23 58 0.18 39 n/a n/a 10 28 58 0.16 43 30 20 48 0.12 34 661161 0.5 30 47 0.13 35 GalNAc₃-3a A_(d) 1.5 23 53 0.14 37 5 26 48 0.15 39 15 32 57 0.15 42 666904 0.5 24 73 0.13 36 GalNAc₃-3a PO 1.5 21 48 0.12 32 5 19 49 0.14 33 15 20 52 0.15 26 675441 0.5 42 148 0.21 36 GalNAc₃-17a A_(d) 1.5 60 95 0.16 34 5 27 75 0.14 37 15 24 61 0.14 36 675442 0.5 26 65 0.15 37 GalNAc₃-18a A_(d) 1.5 25 64 0.15 43 5 27 69 0.15 37 15 30 84 0.14 37

Example 75: Pharmacokinetic Analysis of Oligonucleotides Comprising a 5′-Conjugate Group

The PK of the ASOs in Tables 54, 57 and 60 above was evaluated using liver samples that were obtained following the treatment procedures described in Examples 65, 66, and 74. The liver samples were minced and extracted using standard protocols and analyzed by IP-HPLC-MS alongside an internal standard. The combined tissue level (μg/g) of all metabolites was measured by integrating the appropriate UV peaks, and the tissue level of the full-length ASO missing the conjugate (“parent,” which is Isis No. 353382 in this case) was measured using the appropriate extracted ion chromatograms (EIC).

TABLE 63 PK Analysis in Liver Parent ASO Total Tissue Tissue Level Dosage Level by UV by EIC GalNAc₃ ISIS No. (mg/kg) (μg/g) (μg/g) Cluster CM 353382 3 8.9 8.6 n/a n/a 10 22.4 21.0 30 54.2 44.2 661161 5 32.4 20.7 GalNAc₃-3a A_(d) 15 63.2 44.1 671144 5 20.5 19.2 GalNAc₃-12a A_(d) 15 48.6 41.5 670061 5 31.6 28.0 GalNAc₃-13a A_(d) 15 67.6 55.5 671261 5 19.8 16.8 GalNAc₃-14a A_(d) 15 64.7 49.1 671262 5 18.5 7.4 GalNAc₃-15a A_(d) 15 52.3 24.2 670699 5 16.4 10.4 GalNAc₃-3a T_(d) 15 31.5 22.5 670700 5 19.3 10.9 GalNAc₃-3a A_(e) 15 38.1 20.0 670701 5 21.8 8.8 GalNAc₃-3a T_(e) 15 35.2 16.1 671165 5 27.1 26.5 GalNAc₃-13a A_(d) 15 48.3 44.3 666904 5 30.8 24.0 GalNAc₃-3a PO 15 52.6 37.6 675441 5 25.4 19.0 GalNAc₃-17a A_(d) 15 54.2 42.1 675442 5 22.2 20.7 GalNAc₃-18a A_(d) 15 39.6 29.0

The results in Table 63 above show that there were greater liver tissue levels of the oligonucleotides comprising a GalNAc₃ conjugate group than of the parent oligonucleotide that does not comprise a GalNAc₃ conjugate group (ISIS 353382) 72 hours following oligonucleotide administration, particularly when taking into consideration the differences in dosing between the oligonucleotides with and without a GalNAc₃ conjugate group. Furthermore, by 72 hours, 40-98% of each oligonucleotide comprising a GalNAc₃ conjugate group was metabolized to the parent compound, indicating that the GalNAc₃ conjugate groups were cleaved from the oligonucleotides.

Example 76: Preparation of Oligomeric Compound 230 Comprising GalNAc₃-23

Compound 222 is commercially available. 44.48 ml (0.33 mol) of compound 222 was treated with tosyl chloride (25.39 g, 0.13 mol) in pyridine (500 mL) for 16 hours. The reaction was then evaporated to an oil, dissolved in EtOAc and washed with water, sat. NaHCO₃, brine, and dried over Na₂SO₄. The ethyl acetate was concentrated to dryness and purified by column chromatography, eluted with EtOAc/hexanes (1:1) followed by 10% methanol in CH₂Cl₂ to give compound 223 as a colorless oil. LCMS and NMR were consistent with the structure. 10 g (32.86 mmol) of 1-Tosyltriethylene glycol (compound 223) was treated with sodium azide (10.68 g, 164.28 mmol) in DMSO (100 mL) at room temperature for 17 hours. The reaction mixture was then poured onto water, and extracted with EtOAc. The organic layer was washed with water three times and dried over Na₂SO₄. The organic layer was concentrated to dryness to give 5.3 g of compound 224 (92%). LCMS and NMR were consistent with the structure. 1-Azidotriethylene glycol (compound 224, 5.53 g, 23.69 mmol) and compound 4 (6 g, 18.22 mmol) were treated with 4 A molecular sieves (5 g), and TMSOTf (1.65 ml, 9.11 mmol) in dichloromethane (100 mL) under an inert atmosphere. After 14 hours, the reaction was filtered to remove the sieves, and the organic layer was washed with sat. NaHCO₃, water, brine, and dried over Na₂SO₄. The organic layer was concentrated to dryness and purified by column chromatography, eluted with a gradient of 2 to 4% methanol in dichloromethane to give compound 225. LCMS and NMR were consistent with the structure. Compound 225 (11.9 g, 23.59 mmol) was hydrogenated in EtOAc/Methanol (4:1, 250 mL) over Pearlman's catalyst. After 8 hours, the catalyst was removed by filtration and the solvents removed to dryness to give compound 226. LCMS and NMR were consistent with the structure.

In order to generate compound 227, a solution of nitromethanetrispropionic acid (4.17 g, 15.04 mmol) and Hunig's base (10.3 ml, 60.17 mmol) in DMF (100 mL) were treated dropwise with pentaflourotrifluoro acetate (9.05 ml, 52.65 mmol). After 30 minutes, the reaction was poured onto ice water and extracted with EtOAc. The organic layer was washed with water, brine, and dried over Na₂SO₄. The organic layer was concentrated to dryness and then recrystallized from heptane to give compound 227 as a white solid. LCMS and NMR were consistent with the structure. Compound 227 (1.5 g, 1.93 mmol) and compound 226 (3.7 g, 7.74 mmol) were stirred at room temperature in acetonitrile (15 mL) for 2 hours. The reaction was then evaporated to dryness and purified by column chromatography, eluting with a gradient of 2 to 10% methanol in dichloromethane to give compound 228. LCMS and NMR were consistent with the structure. Compound 228 (1.7 g, 1.02 mmol) was treated with Raney Nickel (about 2 g wet) in ethanol (100 mL) in an atmosphere of hydrogen. After 12 hours, the catalyst was removed by filtration and the organic layer was evaporated to a solid that was used directly in the next step. LCMS and NMR were consistent with the structure. This solid (0.87 g, 0.53 mmol) was treated with benzylglutaric acid (0.18 g, 0.8 mmol), HBTU (0.3 g, 0.8 mmol) and DIEA (273.7 μl, 1.6 mmol) in DMF (5 mL). After 16 hours, the DMF was removed under reduced pressure at 65° C. to an oil, and the oil was dissolved in dichloromethane. The organic layer was washed with sat. NaHCO₃, brine, and dried over Na₂SO₄. After evaporation of the organic layer, the compound was purified by column chromatography and eluted with a gradient of 2 to 20% methanol in dichloromethane to give the coupled product. LCMS and NMR were consistent with the structure. The benzyl ester was deprotected with Pearlman's catalyst under a hydrogen atmosphere for 1 hour. The catalyst was them removed by filtration and the solvents removed to dryness to give the acid. LCMS and NMR were consistent with the structure. The acid (486 mg, 0.27 mmol) was dissolved in dry DMF (3 mL). Pyridine (53.61 μl, 0.66 mmol) was added and the reaction was purged with argon. Pentaflourotriflouro acetate (46.39 μl, 0.4 mmol) was slowly added to the reaction mixture. The color of the reaction changed from pale yellow to burgundy, and gave off a light smoke which was blown away with a stream of argon. The reaction was allowed to stir at room temperature for one hour (completion of reaction was confirmed by LCMS). The solvent was removed under reduced pressure (rotovap) at 70° C. The residue was diluted with DCM and washed with 1N NaHSO₄, brine, saturated sodium bicarbonate and brine again. The organics were dried over Na₂SO₄, filtered, and were concentrated to dryness to give 225 mg of compound 229 as a brittle yellow foam. LCMS and NMR were consistent with the structure.

Oligomeric compound 230, comprising a GalNAc₃-23 conjugate group, was prepared from compound 229 using the general procedure illustrated in Example 46. The GalNAc₃ cluster portion of the GalNAc₃-23 conjugate group (GalNAc₃-23_(a)) can be combined with any cleavable moiety to provide a variety of conjugate groups. The structure of GalNAc₃-23 (GalNAc₃-23_(a)-CM) is shown below:

Example 77: Antisense Inhibition In Vivo by Oligonucleotides Targeting SRB-1 Comprising a GalNAc₃ Conjugate

The oligonucleotides listed below were tested in a dose-dependent study for antisense inhibition of SRB-1 in mice.

TABLE 64 Modified ASOs targeting SRB-1 ISIS GalNAc₃ SEQ No. Sequences (5′ to 3′) Cluster CM ID No. 661161 GalNAc ₃ -3 _(a) - _(o′) A _(do)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds) GalNAc₃-3a A_(d) 4888 G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) 666904 GalNAc ₃ -3 _(a) - _(o′)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds) GalNAc₃-3a PO 4886 G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) 673502 GalNAc ₃ -10 _(a) - _(o′) A _(do)G_(es) ^(m)C_(eo)T_(eo)T_(eo) ^(m)C_(eo)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds) GalNAc₃-10a A_(d) 4888 G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(eo) ^(m)C_(eo) ^(m)C_(es)T_(es)T_(e) 677844 GalNAc ₃ -9 _(a) - _(o′) A _(do)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds) GalNAc₃-9a A_(d) 4888 G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) 677843 GalNAc ₃ -23 _(a) - _(o′) A _(do)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds) GalNAc₃-23a A_(d) 4888 G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) 655861 G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) GalNAc₃-1a A_(d) 4887 ^(m)C_(es)T_(es)T_(eo) A _(do′) -GalNAc ₃ -1 _(a) 677841 G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) GalNAc₃-19a A_(d) 4887 ^(m)C_(es)T_(es)T_(eo) A _(do′) -GalNAc ₃ -19 _(a) 677842 G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) GalNAc₃-20a A_(d) 4887 ^(m)C_(es)T_(es)T_(eo) A _(do′) -GalNAc ₃ -20 _(a)

The structure of GalNAc₃-1_(a) was shown previously in Example 9, GalNAc₃-3_(a) was shown in Example 39, GalNAc₃-9a was shown in Example 52, GalNAc₃-10a was shown in Example 46, GalNAc₃-19_(a) was shown in Example 70, GalNAc₃-20_(a) was shown in Example 71, and GalNAc₃-23_(a) was shown in Example 76.

Treatment

Six to eight week old C57BL/6 mice (Jackson Laboratory, Bar Harbor, Me.) were each injected subcutaneously once at a dosage shown below with an oligonucleotide listed in Table 64 or with saline. Each treatment group consisted of 4 animals. The mice were sacrificed 72 hours following the final administration to determine the SRB-1 mRNA levels using real-time PCR and RIBOGREEN® RNA quantification reagent (Molecular Probes, Inc. Eugene, Oreg.) according to standard protocols. The results below are presented as the average percent of SRB-1 mRNA levels for each treatment group, normalized to the saline control.

As illustrated in Table 65, treatment with antisense oligonucleotides lowered SRB-1 mRNA levels in a dose-dependent manner.

TABLE 65 SRB-1 mRNA (% Saline) Dosage SRB-1 mRNA GalNAc₃ ISIS No. (mg/kg) (% Saline) Cluster CM Saline n/a 100.0 n/a n/a 661161 0.5 89.18 GalNAc₃-3a A_(d) 1.5 77.02 5 29.10 15 12.64 666904 0.5 93.11 GalNAc₃-3a PO 1.5 55.85 5 21.29 15 13.43 673502 0.5 77.75 GalNAc₃-10a A_(d) 1.5 41.05 5 19.27 15 14.41 677844 0.5 87.65 GalNAc₃-9a A_(d) 1.5 93.04 5 40.77 15 16.95 677843 0.5 102.28 GalNAc₃-23a A_(d) 1.5 70.51 5 30.68 15 13.26 655861 0.5 79.72 GalNAc₃-1a A_(d) 1.5 55.48 5 26.99 15 17.58 677841 0.5 67.43 GalNAc₃-19a A_(d) 1.5 45.13 5 27.02 15 12.41 677842 0.5 64.13 GalNAc₃-20a A_(d) 1.5 53.56 5 20.47 15 10.23

Liver transaminase levels, alanine aminotransferase (ALT) and aspartate aminotransferase (AST), in serum were also measured using standard protocols. Total bilirubin and BUN were also evaluated. Changes in body weights were evaluated, with no significant change from the saline group (data not shown). ALTs, ASTs, total bilirubin and BUN values are shown in Table 66 below.

TABLE 66 Total Dosage ALT AST Bilirubin BUN GalNAc₃ ISIS No. (mg/kg) (U/L) (U/L) (mg/dL) (mg/dL) Cluster CM Saline n/a 21 45 0.13 34 n/a n/a 661161 0.5 28 51 0.14 39 GalNAc₃-3a A_(d) 1.5 23 42 0.13 39 5 22 59 0.13 37 15 21 56 0.15 35 666904 0.5 24 56 0.14 37 GalNAc₃-3a PO 1.5 26 68 0.15 35 5 23 77 0.14 34 15 24 60 0.13 35 673502 0.5 24 59 0.16 34 GalNAc₃-10a A_(d) 1.5 20 46 0.17 32 5 24 45 0.12 31 15 24 47 0.13 34 677844 0.5 25 61 0.14 37 GalNAc₃-9a A_(d) 1.5 23 64 0.17 33 5 25 58 0.13 35 15 22 65 0.14 34 677843 0.5 53 53 0.13 35 GalNAc₃-23a A_(d) 1.5 25 54 0.13 34 5 21 60 0.15 34 15 22 43 0.12 38 655861 0.5 21 48 0.15 33 GalNAc₃-1a A_(d) 1.5 28 54 0.12 35 5 22 60 0.13 36 15 21 55 0.17 30 677841 0.5 32 54 0.13 34 GalNAc₃-19a A_(d) 1.5 24 56 0.14 34 5 23 92 0.18 31 15 24 58 0.15 31 677842 0.5 23 61 0.15 35 GalNAc₃-20a A_(d) 1.5 24 57 0.14 34 5 41 62 0.15 35 15 24 37 0.14 32

Example 78: Antisense Inhibition In Vivo by Oligonucleotides Targeting Angiotensinogen Comprising a GalNAc₃ Conjugate

The oligonucleotides listed below were tested in a dose-dependent study for antisense inhibition of Angiotensinogen (AGT) in normotensive Sprague Dawley rats.

TABLE 67 Modified ASOs targeting AGT ISIS GalNAc₃ SEQ No. Sequences (5′ to 3′) Cluster CM ID No. 552668 ^(m)C_(es)A_(es) ^(m)C_(es)T_(es)G_(es)A_(ds) n/a n/a 4892 T_(ds)T_(ds)T_(ds)T_(ds)T_(ds)G_(ds) ^(m)C_(ds) ^(m)C_(ds) ^(m)C_(ds)A_(es)G_(es)G_(es) A_(es)T_(e) 669509 ^(m)C_(es)A_(es) ^(m)C_(es)T_(es)G_(es)A_(ds) GalNAc₃-1_(a) A_(d) 4893 T_(ds)T_(ds)T_(ds)T_(ds)T_(ds)G_(ds) ^(m)C_(ds) ^(m)C_(ds) ^(m)C_(ds)A_(es)G_(es)G_(es) A_(es)T_(eo) A _(do′) -GalNAc ₃ -1 _(a)

The structure of GalNAc₃-1a was shown previously in Example 9.

Treatment

Six week old, male Sprague Dawley rats were each injected subcutaneously once per week at a dosage shown below, for a total of three doses, with an oligonucleotide listed in Table 67 or with PBS. Each treatment group consisted of 4 animals. The rats were sacrificed 72 hours following the final dose. AGT liver mRNA levels were measured using real-time PCR and RIBOGREEN® RNA quantification reagent (Molecular Probes, Inc. Eugene, Oreg.) according to standard protocols. AGT plasma protein levels were measured using the Total Angiotensinogen ELISA (Catalog #JP27412, IBL International, Toronto, ON) with plasma diluted 1:20,000. The results below are presented as the average percent of AGT mRNA levels in liver or AGT protein levels in plasma for each treatment group, normalized to the PBS control.

As illustrated in Table 68, treatment with antisense oligonucleotides lowered AGT liver mRNA and plasma protein levels in a dose-dependent manner, and the oligonucleotide comprising a GalNAc conjugate was significantly more potent than the parent oligonucleotide lacking a GalNAc conjugate.

TABLE 68 AGT liver mRNA and plasma protein levels AGT AGT liver plasma Dosage mRNA protein GalNAc₃ ISIS No. (mg/kg) (% PBS) (% PBS) Cluster CM PBS n/a 100 100 n/a n/a 552668 3 95 122 n/a n/a 10 85 97 30 46 79 90 8 11 669509 0.3 95 70 GalNAc₃-1a A_(d) 1 95 129 3 62 97 10 9 23

Liver transaminase levels, alanine aminotransferase (ALT) and aspartate aminotransferase (AST), in plasma and body weights were also measured at time of sacrifice using standard protocols. The results are shown in Table 69 below.

TABLE 69 Liver transaminase levels and rat body weights Body Dosage ALT AST Weight GalNAc₃ ISIS No. (mg/kg) (U/L) (U/L) (% of baseline) Cluster CM PBS n/a 51 81 186 n/a n/a 552668 3 54 93 183 n/a n/a 10 51 93 194 30 59 99 182 90 56 78 170 669509 0.3 53 90 190 GalNAc₃-1a A_(d) 1 51 93 192 3 48 85 189 10 56 95 189

Example 79: Duration of Action In Vivo of Oligonucleotides Targeting APOC-III Comprising a GalNAc₃ Conjugate

The oligonucleotides listed in Table 70 below were tested in a single dose study for duration of action in mice.

TABLE 70 Modified ASOs targeting APOC-III ISIS GalNAc₃ SEQ No. Sequences (5′ to 3′) Cluster CM ID No. 304801 A_(es)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(ds)T_(ds)T_(ds)G_(ds)T_(ds) ^(m)C_(ds) ^(m)C_(ds)A_(ds)G_(ds) ^(m)C_(ds)T_(es)T_(es) n/a n/a 4878 T_(es)A_(es)T_(e) 647535 A_(es)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(ds)T_(ds)T_(ds)G_(ds)T_(ds) ^(m)C_(ds) ^(m)C_(ds)A_(ds)G_(ds) ^(m)C_(ds)T_(es)T_(es) GalNAc₃-1a A_(d) 4879 T_(es)A_(es)T_(eo) A _(do′) -GalNAc ₃ -1 _(a) 663083 GalNAc ₃ -3 _(a) - _(o′) A _(do)A_(es)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(ds)T_(ds)T_(ds)G_(ds)T_(ds) ^(m)C_(ds) GalNAc₃-3a A_(d) 4894 ^(m)C_(ds)A_(ds)G_(ds) ^(m)C_(ds)T_(es)T_(es)T_(es)A_(es)T_(e) 674449 GalNAC ₃ -7 _(a) - _(o′) A _(do)A_(es)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(ds)T_(ds)T_(ds)G_(ds)T_(ds) ^(m)C_(ds) GalNAc₃-7a A_(d) 4894 ^(m)C_(ds)A_(ds)G_(ds) ^(m)C_(ds)T_(es)T_(es)T_(es)A_(es)T_(e) 674450 GalNAc ₃ -10 _(a) - _(o′) A _(do)A_(es)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(ds)T_(ds)T_(ds)G_(ds)T_(ds) ^(m)C_(ds) GalNAc₃-10a A_(d) 4894 ^(m)C_(ds)A_(ds)G_(ds) ^(m)C_(ds)T_(es)T_(es)T_(es)A_(es)T_(e) 674451 GalNAc ₃ -13 _(a) - _(o′) A _(do)A_(es)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(ds)T_(ds)T_(ds)G_(ds)T_(ds) ^(m)C_(ds) GalNAc₃-13a A_(d) 4894 ^(m)C_(ds)A_(ds)G_(ds) ^(m)C_(ds)T_(es)T_(es)T_(es)A_(es)T_(e) The structure of GalNAc₃-1_(a) was shown previously in Example 9, GalNAc₃-3_(a) was shown in Example 39, GalNAc₃-7_(a) was shown in Example 48, GalNAc₃-10_(a) was shown in Example 46, and GalNAc₃-13_(a) was shown in Example 62. Treatment

Six to eight week old transgenic mice that express human APOC-III were each injected subcutaneously once with an oligonucleotide listed in Table 70 or with PBS. Each treatment group consisted of 3 animals. Blood was drawn before dosing to determine baseline and at 72 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, and 6 weeks following the dose. Plasma triglyceride and APOC-III protein levels were measured as described in Example 20. The results below are presented as the average percent of plasma triglyceride and APOC-III levels for each treatment group, normalized to baseline levels, showing that the oligonucleotides comprising a GalNAc conjugate group exhibited a longer duration of action than the parent oligonucleotide without a conjugate group (ISIS 304801) even though the dosage of the parent was three times the dosage of the oligonucleotides comprising a GalNAc conjugate group.

TABLE 71 Plasma triglyceride and APOC-III protein levels in transgenic mice Time point APOC-III Dosage (days post- Triglycerides protein GalNAc₃ ISIS No. (mg/kg) dose) (% baseline) (% baseline) Cluster CM PBS n/a 3 97 102 n/a n/a 7 101 98 14 108 98 21 107 107 28 94 91 35 88 90 42 91 105 304801 30 3 40 34 n/a n/a 7 41 37 14 50 57 21 50 50 28 57 73 35 68 70 42 75 93 647535 10 3 36 37 GalNAc₃-1a A_(d) 7 39 47 14 40 45 21 41 41 28 42 62 35 69 69 42 85 102 663083 10 3 24 18 GalNAc₃-3a A_(d) 7 28 23 14 25 27 21 28 28 28 37 44 35 55 57 42 60 78 674449 10 3 29 26 GalNAc₃-7a A_(d) 7 32 31 14 38 41 21 44 44 28 53 63 35 69 77 42 78 99 674450 10 3 33 30 GalNAc₃-10a A_(d) 7 35 34 14 31 34 21 44 44 28 56 61 35 68 70 42 83 95 674451 10 3 35 33 GalNAc₃-13a A_(d) 7 24 32 14 40 34 21 48 48 28 54 67 35 65 75 42 74 97

Example 80: Antisense Inhibition In Vivo by Oligonucleotides Targeting Alpha-1 Antitrypsin (A1AT) Comprising a GalNAc₃ Conjugate

The oligonucleotides listed in Table 72 below were tested in a study for dose-dependent inhibition of A1AT in mice.

TABLE 72 Modified ASOs targeting A1AT ISIS GalNAc₃ SEQ ID No. Sequences (5′ to 3′) Cluster CM No. 476366 A_(es) ^(m)C_(es) ^(m)C_(es) ^(m)C_(es)A_(es)A_(ds)T_(ds)T_(ds) ^(m)C_(ds)A_(ds)G_(ds)A_(ds)A_(ds)G_(ds)G_(ds)A_(es)A_(es) n/a n/a 4895 G_(es)G_(es)A_(e) 656326 A_(es) ^(m)C_(es) ^(m)C_(es) ^(m)C_(es)A_(es)A_(ds)T_(ds)T_(ds) ^(m)C_(ds)A_(ds)G_(ds)A_(ds)A_(ds)G_(ds)G_(ds)A_(es)A_(es) GalNAc₃-1a A_(d) 4896 G_(es)G_(es)A_(eo) A _(do′) -GalNAc ₃ -1 _(a) 678381 GalNAc ₃ -3 _(a) - _(o′) A _(do)A_(es) ^(m)C_(es) ^(m)C_(es) ^(m)C_(es)A_(es)A_(ds)T_(ds)T_(ds) ^(m)C_(ds)A_(ds)G_(ds)A_(ds) GalNAc₃-3a A_(d) 4897 A_(ds)G_(ds)G_(ds)A_(es)A_(es)G_(es)G_(es)A_(e) 678382 GalNAc ₃ -7 _(a) - _(o′) A _(do)A_(es) ^(m)C_(es) ^(m)C_(es) ^(m)C_(es)A_(es)A_(ds)T_(ds)T_(ds) ^(m)C_(ds)A_(ds)G_(ds)A_(ds) GalNAc₃-7a A_(d) 4897 A_(ds)G_(ds)G_(ds)A_(es)A_(es)G_(es)G_(es)A_(e) 678383 GalNAc ₃ -10 _(a) - _(o′) A _(do)A_(es) ^(m)C_(es) ^(m)C_(es) ^(m)C_(es)A_(es)A_(ds)T_(ds)T_(ds) ^(m)C_(ds)A_(ds)G_(ds) GalNAc₃-10a A_(d) 4897 A_(ds)A_(ds)G_(ds)G_(ds)A_(es)A_(es)G_(es)G_(es)A_(e) 678384 GalNAc ₃ -13 _(a) - _(o′) A _(do)A_(es) ^(m)C_(es) ^(m)C_(es) ^(m)C_(es)A_(es)A_(ds)T_(ds)T_(ds) ^(m)C_(ds)A_(ds)G_(ds) GalNAc₃-13a A_(d) 4897 A_(ds)A_(ds)G_(ds)G_(ds)A_(es)A_(es)G_(es)G_(es)A_(e)

The structure of GalNAc₃-1_(a) was shown previously in Example 9, GalNAc₃-3_(a) was shown in Example 39, GalNAc₃-7_(a) was shown in Example 48, GalNAc₃-10_(a) was shown in Example 46, and GalNAc₃-13_(a) was shown in Example 62.

Treatment

Six week old, male C57BL/6 mice (Jackson Laboratory, Bar Harbor, Me.) were each injected subcutaneously once per week at a dosage shown below, for a total of three doses, with an oligonucleotide listed in Table 72 or with PBS. Each treatment group consisted of 4 animals. The mice were sacrificed 72 hours following the final administration. A1AT liver mRNA levels were determined using real-time PCR and RIBOGREEN RNA quantification reagent (Molecular Probes, Inc. Eugene, Oreg.) according to standard protocols. A1AT plasma protein levels were determined using the Mouse Alpha 1-Antitrypsin ELISA (catalog #41-A1AMS-E01, Alpco, Salem, N.H.). The results below are presented as the average percent of A1AT liver mRNA and plasma protein levels for each treatment group, normalized to the PBS control.

As illustrated in Table 73, treatment with antisense oligonucleotides lowered A1AT liver mRNA and A1AT plasma protein levels in a dose-dependent manner. The oligonucleotides comprising a GalNAc conjugate were significantly more potent than the parent (ISIS 476366).

TABLE 73 A1AT liver mRNA and plasma protein levels A1AT A1AT liver plasma Dosage mRNA protein GalNAc₃ ISIS No. (mg/kg) (% PBS) (% PBS) Cluster CM PBS n/a 100 100 n/a n/a 476366 5 86 78 n/a n/a 15 73 61 45 30 38 656326 0.6 99 90 GalNAc₃-1a A_(d) 2 61 70 6 15 30 18 6 10 678381 0.6 105 90 GalNAc₃-3a A_(d) 2 53 60 6 16 20 18 7 13 678382 0.6 90 79 GalNAc₃-7a A_(d) 2 49 57 6 21 27 18 8 11 678383 0.6 94 84 GalNAc₃-10a A_(d) 2 44 53 6 13 24 18 6 10 678384 0.6 106 91 GalNAc₃-13a A_(d) 2 65 59 6 26 31 18 11 15

Liver transaminase and BUN levels in plasma were measured at time of sacrifice using standard protocols. Body weights and organ weights were also measured. The results are shown in Table 74 below. Body weight is shown as % relative to baseline. Organ weights are shown as % of body weight relative to the PBS control group.

TABLE 74 Body Liver Kidney Spleen Dosage ALT AST BUN weight weight weight weight ISIS No. (mg/kg) (U/L) (U/L) (mg/dL) (% baseline) (Rel % BW) (Rel % BW) (Rel % BW) PBS n/a 25 51 37 119 100 100 100 476366 5 34 68 35 116 91 98 106 15 37 74 30 122 92 101 128 45 30 47 31 118 99 108 123 656326 0.6 29 57 40 123 100 103 119 2 36 75 39 114 98 111 106 6 32 67 39 125 99 97 122 18 46 77 36 116 102 109 101 678381 0.6 26 57 32 117 93 109 110 2 26 52 33 121 96 106 125 6 40 78 32 124 92 106 126 18 31 54 28 118 94 103 120 678382 0.6 26 42 35 114 100 103 103 2 25 50 31 117 91 104 117 6 30 79 29 117 89 102 107 18 65 112 31 120 89 104 113 678383 0.6 30 67 38 121 91 100 123 2 33 53 33 118 98 102 121 6 32 63 32 117 97 105 105 18 36 68 31 118 99 103 108 678384 0.6 36 63 31 118 98 103 98 2 32 61 32 119 93 102 114 6 34 69 34 122 100 100 96 18 28 54 30 117 98 101 104

Example 81: Duration of Action In Vivo of Oligonucleotides Targeting A1AT Comprising a GalNAc₃ Cluster

The oligonucleotides listed in Table 72 were tested in a single dose study for duration of action in mice.

Treatment

Six week old, male C57BL/6 mice were each injected subcutaneously once with an oligonucleotide listed in Table 72 or with PBS. Each treatment group consisted of 4 animals. Blood was drawn the day before dosing to determine baseline and at 5, 12, 19, and 25 days following the dose. Plasma A1AT protein levels were measured via ELISA (see Example 80). The results below are presented as the average percent of plasma A1AT protein levels for each treatment group, normalized to baseline levels. The results show that the oligonucleotides comprising a GalNAc conjugate were more potent and had longer duration of action than the parent lacking a GalNAc conjugate (ISIS 476366). Furthermore, the oligonucleotides comprising a 5′-GalNAc conjugate (ISIS 678381, 678382, 678383, and 678384) were generally even more potent with even longer duration of action than the oligonucleotide comprising a 3′-GalNAc conjugate (ISIS 656326).

TABLE 75 Plasma A1AT protein levels in mice Time point Dosage (days post- A1AT GalNAc₃ ISIS No. (mg/kg) dose) (% baseline) Cluster CM PBS n/a 5 93 n/a n/a 12 93 19 90 25 97 476366 100 5 38 n/a n/a 12 46 19 62 25 77 656326 18 5 33 GalNAc₃-1a A_(d) 12 36 19 51 25 72 678381 18 5 21 GalNAc₃-3a A_(d) 12 21 19 35 25 48 678382 18 5 21 GalNAc₃-7a A_(d) 12 21 19 39 25 60 678383 18 5 24 GalNAc₃-10a A_(d) 12 21 19 45 25 73 678384 18 5 29 GalNAc₃-13a A_(d) 12 34 19 57 25 76

Example 82: Antisense Inhibition In Vitro by Oligonucleotides Targeting SRB-1 Comprising a GalNAc₃ Conjugate

Primary mouse liver hepatocytes were seeded in 96 well plates at 15,000 cells/well 2 hours prior to treatment. The oligonucleotides listed in Table 76 were added at 2, 10, 50, or 250 nM in Williams E medium and cells were incubated overnight at 37° C. in 5% CO₂. Cells were lysed 16 hours following oligonucleotide addition, and total RNA was purified using RNease 3000 BioRobot (Qiagen). SRB-1 mRNA levels were determined using real-time PCR and RIBOGREEN RNA quantification reagent (Molecular Probes, Inc. Eugene, Oreg.) according to standard protocols. IC₅₀ values were determined using Prism 4 software (GraphPad). The results show that oligonucleotides comprising a variety of different GalNAc conjugate groups and a variety of different cleavable moieties are significantly more potent in an in vitro free uptake experiment than the parent oligonucleotides lacking a GalNAc conjugate group (ISIS 353382 and 666841).

TABLE 76 Inhibition of SRB-1 expression in vitro ISIS GalNAc IC₅₀ SEQ No. Sequence (5′ to 3′) Linkages cluster CM (nM) ID No. 353382 G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) PS n/a n/a 250 4886 ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) 655861 G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) PS GalNAc₃- A_(d) 40 4887 ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(eo) A _(do′) -GalNAC ₃ -1a 1_(a) 661161 GalNAc ₃ -3 _(a) - _(o′) A _(do)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) PS GalNAc₃- A_(d) 40 4888 ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) 3_(a) 661162 GalNAc ₃ -3 _(a) - _(o′) A _(do)G_(es) ^(m)C_(eo)T_(eo)T_(eo) ^(m)C_(eo)A_(ds)G_(ds)T_(ds) PO/PS GalNAc₃- A_(d) 8 4888 ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(eo) ^(m)C_(eo) ^(m)C_(es)T_(es)T_(e) 3_(a) 664078 G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) PS GalNAc₃- A_(d) 20 4887 ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(eo) A _(do′) -GalNAc ₃ -9 _(a) 9_(a) 665001 GalNAc ₃ -8 _(a) - _(o′) A _(do)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) PS GalNAc₃- A_(d) 70 4888 ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) 8_(a) 666224 GalNAc ₃ -5 _(a) - _(o′) A _(do)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) PS GalNAc₃- A_(d) 80 4888 ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) 5_(a) 666841 G_(es) ^(m)C_(eo)T_(eo)T_(eo) ^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) PO/PS n/a n/a >250 4886 ^(m)C_(ds)T_(ds)T_(eo) ^(m)C_(eo) ^(m)C_(es)T_(es)T_(e) 666881 GalNAc ₃ -10 _(a) - _(o′) A _(do)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) PS GalNAc₃- A_(d) 30 4888 ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) 10_(a) 666904 GalNAc ₃ -3 _(a) - _(o′)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds) PS GalNAc₃- PO 9 4886 A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds T) _(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) 3_(a) 666924 GalNAc ₃ -3 _(a) - _(o′) T _(do)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) PS GalNAc₃- T_(d) 15 4891 ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) 3_(a) 666961 GalNAC ₃ -6 _(a) - _(o′) A _(do)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) PS GalNAc₃- A_(d) 150 4888 ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) 6_(a) 666981 GalNAC ₃ -7 _(a) - _(o′) A _(do)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) PS GalNAc₃- A_(d) 20 4888 ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) 7_(a) 670061 GalNAC ₃ -13 _(a) - _(o′) A _(do)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) PS GalNAc₃- A_(d) 30 4888 ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) 13_(a) 670699 GalNAC ₃ -3 _(a) - _(o′) T _(do)G_(es) ^(m)C_(eo)T_(eo)T_(eo) ^(m)C_(eo)A_(ds)G_(ds)T_(ds) PO/PS GalNAc₃- T_(d) 30 4891 ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(eo) ^(m)C_(eo) ^(m)C_(es)T_(es)T_(e) 3_(a) 670700 GalNAC ₃ -3 _(a) - _(o′) A _(eo)G_(es) ^(m)C_(eo)T_(eo)T_(eo) ^(m)C_(eo)A_(ds)G_(ds)T_(ds) PO/PS GalNAc₃- A_(e) 30 4888 ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(eo) ^(m)C_(eo) ^(m)C_(es)T_(es)T 3_(a) 670701 GalNAC ₃ -3 _(a) - _(o′) T _(eo)G_(es) ^(m)C_(eo)T_(eo)T_(eo) ^(m)C_(eo)A_(ds)G_(ds)T_(ds) PO/PS GalNAc₃- T_(e) 25 4891 ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(eo) ^(m)C_(eo) ^(m)C_(es)T_(es)T_(e) 3_(a) 671144 GalNAC ₃ -12 _(a) - _(o′) A _(do)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) PS GalNAc₃- A_(d) 30 4888 ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) 12_(a) 671165 GalNAC ₃ -13 _(a) - _(o′) A _(do)G_(es) ^(m)C_(eo)T_(eo)T_(eo) ^(m)C_(eo)A_(ds)G_(ds)T_(ds) PO/PS GalNAc₃- A_(d) 8 4888 ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(eo) ^(m)C_(eo) ^(m)C_(es)T_(es)T 13_(a) 671261 GalNAC ₃ -14 _(a) - _(o′) A _(do)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) PS GalNAc₃- A_(d) >250 4888 ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) 14_(a) 671262 GalNAC ₃ -15 _(a) - _(o′) A _(do)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) PS GalNAc₃- A_(d) >250 4888 ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) 15_(a) 673501 GalNAC ₃ -7 _(a) - _(o′) A _(do)G_(es) ^(m)C_(eo)T_(eo)T_(eo) ^(m)C_(eo)A_(ds)G_(ds)T_(ds) PO/PS GalNAc₃- A_(d) 30 4888 ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(eo) ^(m)C_(eo) ^(m)C_(es)T_(es)T_(e) 7_(a) 673502 GalNAC ₃ -10 _(a) - _(o′) A _(do)G_(es) ^(m)C_(eo)T_(eo)T_(eo) ^(m)C_(eo)A_(ds)G_(ds)T_(ds) PO/PS GalNAc₃- A_(d) 8 4888 ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(eo) ^(m)C_(eo) ^(m)C_(es)T_(es)T_(e) 10_(a) 675441 GalNAC ₃ -17 _(a) - _(o′) A _(do)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) PS GalNAc₃- A_(d) 30 4888 ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) 17_(a) 675442 GalNAC ₃ -18 _(a) - _(o′) A _(do)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) PS GalNAc₃- A_(d) 20 4888 ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) 18_(a) 677841 G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) PS GalNAc₃- A_(d) 40 4887 ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(eo) A _(do′) -GalNAC ₃ -19 _(a) 19_(a) 677842 G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) PS GalNAc₃- A_(d) 30 4887 ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(eo) A _(do′) -GalNAC ₃ -20 _(a) 20_(a) 677843 GalNAC ₃ -23 _(a) - _(o′) A _(do)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) PS GalNAc₃- A_(d) 40 4888 ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) 23_(a) The structure of GalNAc₃-1_(a) was shown previously in Example 9, GalNAc₃-3_(a) was shown in Example 39, GalNAc₃-5_(a) was shown in Example 49, GalNAc₃-6_(a) was shown in Example 51, GalNAc₃-7_(a) was shown in Example 48, GalNAc₃-8_(a) was shown in Example 47, GalNAc₃-9_(a) was shown in Example 52, GalNAc₃-10_(a) was shown in Example 46, GalNAc₃-12_(a) was shown in Example 61, GalNAc₃-13_(a) was shown in Example 62, GalNAc₃-14_(a) was shown in Example 63, GalNAc₃-15_(a) was shown in Example 64, GalNAc₃-17_(a) was shown in Example 68, GalNAc₃-18_(a) was shown in Example 69, GalNAc₃-19_(a) was shown in Example 70, GalNAc₃-20_(a) was shown in Example 71, and GalNAc₃-23_(a) was shown in Example 76.

Example 83: Antisense Inhibition In Vivo by Oligonucleotides Targeting Factor XI Comprising a GalNAc₃ Cluster

The oligonucleotides listed in Table 77 below were tested in a study for dose-dependent inhibition of Factor XI in mice.

TABLE 77 Modified oligonucleotides targeting Factor XI ISIS GalNAc SEQ No. Sequence (5′ to 3′) cluster CM ID No. 404071 T_(es)G_(es)G_(es)T_(es)A_(es)A_(ds)T_(ds) ^(m)C_(ds) ^(m)C_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(ds)T_(ds) ^(m)C_(ds)A_(es)G_(es) n/a n/a 4889 A_(es)G_(es)Ge 656173 T_(es)G_(eo)G_(eo)T_(eo)A_(eo)A_(ds)T_(ds) ^(m)C_(ds) ^(m)C_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(ds)T_(ds) ^(m)C_(ds)A_(eo)G_(eo) GalNAc₃-1_(a) A_(d) 4890 A_(es)G_(es)G_(eo) A _(do′) -GalNAC ₃ -1 _(a) 663086 GalNAc ₃ -3 _(a) - _(o′) A _(do)T_(es)G_(eo)G_(eo)T_(eo)A_(eo)A_(ds)T_(ds) ^(m)C_(ds) ^(m)C_(ds)A_(ds) ^(m)C_(ds)T_(ds) GalNAc₃-3_(a) A_(d) 4898 T_(ds)T_(ds) ^(m)C_(ds)A_(eo)G_(eo)A_(es)G_(es)G_(e) 678347 GalNAc ₃ -7 _(a) - _(o′) A _(do)T_(es)G_(eo)G_(eo)T_(eo)A_(eo)A_(ds)T_(ds) ^(m)C_(ds) ^(m)C_(ds)A_(ds) ^(m)C_(ds)T_(ds) GalNAc₃-7_(a) A_(d) 4898 T_(ds)T_(ds) ^(m)C_(ds)A_(eo)G_(eo)A_(es)G_(es)G_(e) 678348 GalNAc ₃ -10 _(a) - _(o′) A _(do)T_(es)G_(eo)G_(eo)T_(eo)A_(eo)A_(ds)T_(ds) ^(m)C_(ds) ^(m)C_(ds)A_(ds) ^(m)C_(ds) GalNAc₃-10_(a) A_(d) 4898 T_(ds)T_(ds)T_(ds) ^(m)C_(ds)A_(eo)G_(eo)A_(es)G_(es)G_(e) 678349 GalNAc ₃ -13 _(a) - _(o′) A _(do)T_(es)G_(eo)G_(eo)T_(eo)A_(eo)A_(ds)T_(ds) ^(m)C_(ds) ^(m)C_(ds)A_(ds) ^(m)C_(ds) GalNAc₃-13_(a) A_(d) 4898 T_(ds)T_(ds)T_(ds) ^(m)C_(ds)A_(eo)G_(eo)A_(es)G_(es)G_(e)

The structure of GalNAc₃-1_(a) was shown previously in Example 9, GalNAc₃-3_(a) was shown in Example 39, GalNAc₃-7_(a) was shown in Example 48, GalNAc₃-10_(a) was shown in Example 46, and GalNAc₃-13_(a) was shown in Example 62.

Treatment

Six to eight week old mice were each injected subcutaneously once per week at a dosage shown below, for a total of three doses, with an oligonucleotide listed below or with PBS. Each treatment group consisted of 4 animals. The mice were sacrificed 72 hours following the final dose. Factor XI liver mRNA levels were measured using real-time PCR and normalized to cyclophilin according to standard protocols. Liver transaminases, BUN, and bilirubin were also measured. The results below are presented as the average percent for each treatment group, normalized to the PBS control.

As illustrated in Table 78, treatment with antisense oligonucleotides lowered Factor XI liver mRNA in a dose-dependent manner. The results show that the oligonucleotides comprising a GalNAc conjugate were more potent than the parent lacking a GalNAc conjugate (ISIS 404071). Furthermore, the oligonucleotides comprising a 5′-GalNAc conjugate (ISIS 663086, 678347, 678348, and 678349) were even more potent than the oligonucleotide comprising a 3′-GalNAc conjugate (ISIS 656173).

TABLE 78 Factor XI liver mRNA, liver transaminase, BUN, and bilirubin levels Factor XI Dosage mRNA ALT AST BUN Bilirubin GalNAc₃ SEQ ISIS No. (mg/kg) (% PBS) (U/L) (U/L) (mg/dL) (mg/dL) Cluster ID No. PBS n/a 100 63 70 21 0.18 n/a n/a 404071 3 65 41 58 21 0.15 n/a 4889 10 33 49 53 23 0.15 30 17 43 57 22 0.14 656173 0.7 43 90 89 21 0.16 GalNAc₃-1a 4890 2 9 36 58 26 0.17 6 3 50 63 25 0.15 663086 0.7 33 91 169 25 0.16 GalNAc₃-3a 4898 2 7 38 55 21 0.16 6 1 34 40 23 0.14 678347 0.7 35 28 49 20 0.14 GalNAc₃-7a 4898 2 10 180 149 21 0.18 6 1 44 76 19 0.15 678348 0.7 39 43 54 21 0.16 GalNAc₃-10a 4898 2 5 38 55 22 0.17 6 2 25 38 20 0.14 678349 0.7 34 39 46 20 0.16 GalNAc₃-13a 4898 2 8 43 63 21 0.14 6 2 28 41 20 0.14

Example 84: Duration of Action In Vivo of Oligonucleotides Targeting Factor XI Comprising a GalNAc₃ Conjugate

The oligonucleotides listed in Table 77 were tested in a single dose study for duration of action in mice.

Treatment Six to eight week old mice were each injected subcutaneously once with an oligonucleotide listed in Table 77 or with PBS. Each treatment group consisted of 4 animals. Blood was drawn by tail bleeds the day before dosing to determine baseline and at 3, 10, and 17 days following the dose. Plasma Factor XI protein levels were measured by ELISA using Factor XI capture and biotinylated detection antibodies from R & D Systems, Minneapolis, Minn. (catalog #AF2460 and #BAF2460, respectively) and the OptEIA Reagent Set B (Catalog #550534, BD Biosciences, San Jose, Calif.). The results below are presented as the average percent of plasma Factor XI protein levels for each treatment group, normalized to baseline levels. The results show that the oligonucleotides comprising a GalNAc conjugate were more potent with longer duration of action than the parent lacking a GalNAc conjugate (ISIS 404071). Furthermore, the oligonucleotides comprising a 5′-GalNAc conjugate (ISIS 663086, 678347, 678348, and 678349) were even more potent with an even longer duration of action than the oligonucleotide comprising a 3′-GalNAc conjugate (ISIS 656173).

TABLE 79 Plasma Factor XI protein levels in mice ISIS Dosage Time point (days Factor XI (% SEQ ID No. (mg/kg) post-dose) baseline) GalNAc₃ Cluster CM No. PBS n/a 3 123 n/a n/a n/a 10 56 17 100 404071 30 3 11 n/a n/a 4889 10 47 17 52 656173 6 3 1 GalNAc₃-1a A_(d) 4890 10 3 17 21 663086 6 3 1 GalNAc₃-3a A_(d) 4898 10 2 17 9 678347 6 3 1 GalNAc₃-7a A_(d) 4898 10 1 17 8 678348 6 3 1 GalNAc₃-10a A_(d) 4898 10 1 17 6 678349 6 3 1 GalNAc₃-13a A_(d) 4898 10 1 17 5

Example 85: Antisense Inhibition In Vivo by Oligonucleotides Targeting SRB-1 Comprising a GalNAc₃ Conjugate

Oligonucleotides listed in Table 76 were tested in a dose-dependent study for antisense inhibition of SRB-1 in mice.

Treatment

Six to eight week old C57BL/6 mice were each injected subcutaneously once per week at a dosage shown below, for a total of three doses, with an oligonucleotide listed in Table 76 or with saline. Each treatment group consisted of 4 animals. The mice were sacrificed 48 hours following the final administration to determine the SRB-1 mRNA levels using real-time PCR and RIBOGREEN® RNA quantification reagent (Molecular Probes, Inc. Eugene, Oreg.) according to standard protocols. The results below are presented as the average percent of liver SRB-1 mRNA levels for each treatment group, normalized to the saline control.

As illustrated in Tables 80 and 81, treatment with antisense oligonucleotides lowered SRB-1 mRNA levels in a dose-dependent manner.

TABLE 80 SRB-1 mRNA in liver Dosage SRB-1 mRNA GalNAc₃ ISIS No. (mg/kg) (% Saline) Cluster CM Saline n/a 100 n/a n/a 655861 0.1 94 GalNAc₃-1a A_(d) 0.3 119 1 68 3 32 661161 0.1 120 GalNAc₃-3a A_(d) 0.3 107 1 68 3 26 666881 0.1 107 GalNAc₃-10a A_(d) 0.3 107 1 69 3 27 666981 0.1 120 GalNAc₃-7a A_(d) 0.3 103 1 54 3 21 670061 0.1 118 GalNAc₃-13a A_(d) 0.3 89 1 52 3 18 677842 0.1 119 GalNAc₃-20a A_(d) 0.3 96 1 65 3 23

TABLE 81 SRB-1 mRNA in liver Dosage SRB-1 mRNA GalNAc₃ ISIS No. (mg/kg) (% Saline) Cluster CM 661161 0.1 107 GalNAc₃-3a A_(d) 0.3 95 1 53 3 18 677841 0.1 110 GalNAc₃-19a A_(d) 0.3 88 1 52 3 25

Liver transaminase levels, total bilirubin, BUN, and body weights were also measured using standard protocols. Average values for each treatment group are shown in Table 82 below.

TABLE 82 ISIS Dosage ALT AST Bilirubin BUN Body Weight No. (mg/kg) (U/L) (U/L) (mg/dL) (mg/dL) (% baseline) GalNAc₃ Cluster CM Saline n/a 19 39 0.17 26 118 n/a n/a 655861 0.1 25 47 0.17 27 114 GalNAc₃-1a A_(d) 0.3 29 56 0.15 27 118 1 20 32 0.14 24 112 3 27 54 0.14 24 115 661161 0.1 35 83 0.13 24 113 GalNAc₃-3a A_(d) 0.3 42 61 0.15 23 117 1 34 60 0.18 22 116 3 29 52 0.13 25 117 666881 0.1 30 51 0.15 23 118 GalNAc₃-10a A_(d) 0.3 49 82 0.16 25 119 1 23 45 0.14 24 117 3 20 38 0.15 21 112 666981 0.1 21 41 0.14 22 113 GalNAc₃-7a A_(d) 0.3 29 49 0.16 24 112 1 19 34 0.15 22 111 3 77 78 0.18 25 115 670061 0.1 20 63 0.18 24 111 GalNAc₃-13a A_(d) 0.3 20 57 0.15 21 115 1 20 35 0.14 20 115 3 27 42 0.12 20 116 677842 0.1 20 38 0.17 24 114 GalNAc₃-20a A_(d) 0.3 31 46 0.17 21 117 1 22 34 0.15 21 119 3 41 57 0.14 23 118

Example 86: Antisense Inhibition In Vivo by Oligonucleotides Targeting TTR Comprising a GalNAc₃ Cluster

Oligonucleotides listed in Table 83 below were tested in a dose-dependent study for antisense inhibition of human transthyretin (TTR) in transgenic mice that express the human TTR gene.

Treatment

Eight week old TTR transgenic mice were each injected subcutaneously once per week for three weeks, for a total of three doses, with an oligonucleotide and dosage listed in the tables below or with PBS. Each treatment group consisted of 4 animals. The mice were sacrificed 72 hours following the final administration. Tail bleeds were performed at various time points throughout the experiment, and plasma TTR protein, ALT, and AST levels were measured and reported in Tables 85-87. After the animals were sacrificed, plasma ALT, AST, and human TTR levels were measured, as were body weights, organ weights, and liver human TTR mRNA levels. TTR protein levels were measured using a clinical analyzer (AU480, Beckman Coulter, CA). Real-time PCR and RIBOGREEN RNA quantification reagent (Molecular Probes, Inc. Eugene, Oreg.) were used according to standard protocols to determine liver human TTR mRNA levels. The results presented in Tables 84-87 are the average values for each treatment group. The mRNA levels are the average values relative to the average for the PBS group. Plasma protein levels are the average values relative to the average value for the PBS group at baseline. Body weights are the average percent weight change from baseline until sacrifice for each individual treatment group. Organ weights shown are normalized to the animal's body weight, and the average normalized organ weight for each treatment group is then presented relative to the average normalized organ weight for the PBS group.

In Tables 84-87, “BL” indicates baseline, measurements that were taken just prior to the first dose. As illustrated in Tables 84 and 85, treatment with antisense oligonucleotides lowered TTR expression levels in a dose-dependent manner. The oligonucleotides comprising a GalNAc conjugate were more potent than the parent lacking a GalNAc conjugate (ISIS 420915). Furthermore, the oligonucleotides comprising a GalNAc conjugate and mixed PS/PO internucleoside linkages were even more potent than the oligonucleotide comprising a GalNAc conjugate and full PS linkages.

TABLE 83 Oligonucleotides targeting human TTR GalNAc SEQ Isis No. Sequence 5′ to 3′ Linkages cluster CM ID No. 420915 T_(es) ^(m)C_(es)T_(es)T_(es)G_(es)G_(ds)T_(ds)T_(ds)A_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds)A_(ds) PS n/a n/a 4899 A_(es)T_(es) ^(m)C_(es) ^(m)C_(es) ^(m)C_(e) 660261 T_(es) ^(m)C_(es)T_(es)T_(es)G_(es)G_(ds)T_(ds)T_(ds)A_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds)A_(ds) PS GalNAc₃-1a A_(d) 4900 A_(es)T_(es) ^(m)C_(es) ^(m)C_(es) ^(m)C_(eo) A _(do′) -GalNAc ₃ -1 _(a) 682883 GalNAc ₃ -3 _(a-o′)T_(es) ^(m)C_(eo)T_(eo)T_(eo)G_(eo)G_(ds)T_(ds)T_(ds)A_(ds) ^(m)C_(ds)A_(ds) PS/PO GalNAc₃-3a PO 4899 T_(ds)G_(ds)A_(ds)A_(ds)A_(eo)T_(eo) ^(m)C_(es) ^(m)C_(es) ^(m)C_(e) 682884 GalNAc ₃ -7 _(a-o′)T_(es) ^(m)C_(eo)T_(eo)T_(eo)G_(eo)G_(ds)T_(ds)T_(ds)A_(ds) ^(m)C_(ds)A_(ds) PS/PO GalNAc₃-7a PO 4899 T_(ds)G_(ds)A_(ds)A_(ds)A_(eo)T_(eo) ^(m)C_(es) ^(m)C_(es) ^(m)C_(e) 682885 GalNAc ₃ -10 _(a-o′)T_(es) ^(m)C_(eo)T_(eo)T_(eo)G_(eo)G_(ds)T_(ds)T_(ds)A_(ds) ^(m)C_(ds) PS/PO GalNAc₃-10a PO 4899 A_(ds)T_(ds)G_(ds)A_(ds)A_(ds)A_(eo)T_(eo) ^(m)C_(es) ^(m)C_(es) ^(m)C_(e) 682886 GalNAc ₃ -13 _(a-o′)T_(es) ^(m)C_(eo)T_(eo)T_(eo)Ge_(eo)G_(ds)T_(ds)T_(ds)A_(ds) ^(m)C_(ds) PS/PO GalNAc₃-13a PO 4899 A_(ds)T_(ds)G_(ds)A_(ds)A_(ds)A_(eo)T_(eo) ^(m)C_(es) ^(m)C_(es) ^(m)C_(e) 684057 T_(es) ^(m)C_(eo)T_(eo)T_(eo)G_(eo)G_(ds)T_(ds)T_(ds)A_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds)A_(ds) PS GalNAc₃-19a A_(d) 4900 A_(eo)T_(eo) ^(m)C_(es) ^(m)C_(es) ^(m)C_(eo) A _(do′) -GalNAc ₃ -19 _(a) The legend for Table 85 can be found in Example 74. The structure of GalNAc₃-1 was shown in Example 9. The structure of GalNAc₃-3_(a) was shown in Example 39. The structure of GalNAc₃-7_(a) was shown in Example 48. The structure of GalNAc₃-10_(a) was shown in Example 46. The structure of GalNAc₃-13_(a) was shown in Example 62. The structure of GalNAc₃-19_(a) was shown in Example 70.

TABLE 84 Antisense inhibition of human TTR in vivo Plasma TTR TTR Isis Dosage mRNA protein GalNAc SEQ No. (mg/kg) (% PBS) (% PBS) cluster CM ID No. PBS n/a 100 100 n/a n/a 420915 6 99 95 n/a n/a 4899 20 48 65 60 18 28 660261 0.6 113 87 GalNAc₃-1a A_(d) 4900 2 40 56 6 20 27 20 9 11

TABLE 85 Antisense inhibition of human TTR in vivo TTR Plasma TTR protein (% PBS at BL) SEQ Dosage mRNA Day 17 GalNAc ID Isis No. (mg/kg) (% PBS) BL Day 3 Day 10 (After sac) cluster CM No. PBS n/a 100 100 96 90 114 n/a n/a 420915 6 74 106 86 76 83 n/a n/a 4899 20 43 102 66 61 58 60 24 92 43 29 32 682883 0.6 60 88 73 63 68 GalNAc₃- PO 4899 2 18 75 38 23 23 3a 6 10 80 35 11 9 682884 0.6 56 88 78 63 67 GalNAc₃- PO 4899 2 19 76 44 25 23 7a 6 15 82 35 21 24 682885 0.6 60 92 77 68 76 GalNAc₃- PO 4899 2 22 93 58 32 32 10a 6 17 85 37 25 20 682886 0.6 57 91 70 64 69 GalNAc₃- PO 4899 2 21 89 50 31 30 13a 6 18 102 41 24 27 684057 0.6 53 80 69 56 62 GalNAc₃- A_(d) 4900 2 21 92 55 34 30 19a 6 11 82 50 18 13

TABLE 86 Transaminase levels, body weight changes, and relative organ weights ALT (U/L) AST (U/L) Body Liver Spleen Kidney SEQ Dosage Day Day Day Day (% (% (% (% ID Isis No. (mg/kg) BL Day 3 10 17 BL Day 3 10 17 BL) PBS) PBS) PBS) No. PBS n/a 33 34 33 24 58 62 67 52 105 100 100 100 n/a 420915 6 34 33 27 21 64 59 73 47 115 99 89 91 4899 20 34 30 28 19 64 54 56 42 111 97 83 89 60 34 35 31 24 61 58 71 58 113 102 98 95 660261 0.6 33 38 28 26 70 71 63 59 111 96 99 92 4900 2 29 32 31 34 61 60 68 61 118 100 92 90 6 29 29 28 34 58 59 70 90 114 99 97 95 20 33 32 28 33 64 54 68 95 114 101 106 92

TABLE 87 Transaminase levels, body weight changes, and relative organ weights ALT (U/L) AST (U/L) Body Liver Spleen Kidney SEQ Dosage Day Day Day Day (% (% (% (% ID Isis No. (mg/kg) BL Day 3 10 17 BL Day 3 10 17 BL) PBS) PBS) PBS) No. PBS n/a 32 34 37 41 62 78 76 77 104 100 100 100 n/a 420915 6 32 30 34 34 61 71 72 66 102 103 102 105 4899 20 41 34 37 33 80 76 63 54 106 107 135 101 60 36 30 32 34 58 81 57 60 106 105 104 99 682883 0.6 32 35 38 40 53 81 74 76 104 101 112 95 4899 2 38 39 42 43 71 84 70 77 107 98 116 99 6 35 35 41 38 62 79 103 65 105 103 143 97 682884 0.6 33 32 35 34 70 74 75 67 101 100 130 99 4899 2 31 32 38 38 63 77 66 55 104 103 122 100 6 38 32 36 34 65 85 80 62 99 105 129 95 682885 0.6 39 26 37 35 63 63 77 59 100 109 109 112 4899 2 30 26 38 40 54 56 71 72 102 98 111 102 6 27 27 34 35 46 52 56 64 102 98 113 96 682886 0.6 30 40 34 36 58 87 54 61 104 99 120 101 4899 2 27 26 34 36 51 55 55 69 103 91 105 92 6 40 28 34 37 107 54 61 69 109 100 102 99 684057 0.6 35 26 33 39 56 51 51 69 104 99 110 102 4900 2 33 32 31 40 54 57 56 87 103 100 112 97 6 39 33 35 40 67 52 55 92 98 104 121 108

Example 87: Duration of Action In Vivo by Single Doses of Oligonucleotides Targeting TTR Comprising a GalNAc₃ Cluster

ISIS numbers 420915 and 660261 (see Table 83) were tested in a single dose study for duration of action in mice. ISIS numbers 420915, 682883, and 682885 (see Table 83) were also tested in a single dose study for duration of action in mice.

Treatment

Eight week old, male transgenic mice that express human TTR were each injected subcutaneously once with 100 mg/kg ISIS No. 420915 or 13.5 mg/kg ISIS No. 660261. Each treatment group consisted of 4 animals. Tail bleeds were performed before dosing to determine baseline and at days 3, 7, 10, 17, 24, and 39 following the dose. Plasma TTR protein levels were measured as described in Example 86. The results below are presented as the average percent of plasma TTR levels for each treatment group, normalized to baseline levels.

TABLE 88 Plasma TTR protein levels ISIS Dosage Time point GalNAc₃ No. (mg/kg) (days post-dose) TTR (% baseline) Cluster CM SEQ ID No. 420915 100 3 30 n/a n/a 4899 7 23 10 35 17 53 24 75 39 100 660261 13.5 3 27 GalNAc₃-1a A_(d) 4900 7 21 10 22 17 36 24 48 39 69 Treatment

Female transgenic mice that express human TTR were each injected subcutaneously once with 100 mg/kg ISIS No. 420915, 10.0 mg/kg ISIS No. 682883, or 10.0 mg/kg 682885. Each treatment group consisted of 4 animals. Tail bleeds were performed before dosing to determine baseline and at days 3, 7, 10, 17, 24, and 39 following the dose. Plasma TTR protein levels were measured as described in Example 86. The results below are presented as the average percent of plasma TTR levels for each treatment group, normalized to baseline levels.

TABLE 89 Plasma TTR protein levels ISIS Dosage Time point GalNAc₃ No. (mg/kg) (days post-dose) TTR (% baseline) Cluster CM SEQ ID No. 420915 100 3 48 n/a n/a 4899 7 48 10 48 17 66 31 80 682883 10.0 3 45 GalNAc₃-3a PO 4899 7 37 10 38 17 42 31 65 682885 10.0 3 40 GalNAc₃-10a PO 4899 7 33 10 34 17 40 31 64 The results in Tables 88 and 89 show that the oligonucleotides comprising a GalNAc conjugate are more potent with a longer duration of action than the parent oligonucleotide lacking a conjugate (ISIS 420915).

Example 88: Splicing Modulation In Vivo by Oligonucleotides Targeting SMN Comprising a GalNAc₃ Conjugate

The oligonucleotides listed in Table 90 were tested for splicing modulation of human survival of motor neuron (SMN) in mice.

TABLE 90 Modified ASOs targeting SMN ISIS GalNAc₃ SEQ No. Sequences (5′ to 3′) Cluster CM ID No. 387954 A_(es)T_(es)T_(es) ^(m)C_(es)A_(es) ^(m)C_(es)T_(es)T_(es)T_(es) ^(m)C_(es)A_(es)T_(es)A_(es)A_(es)T_(es)G_(es) ^(m)C_(es)T_(es)G_(es) n/a n/a 4901 G_(e) 699819 GalNAc ₃ -7 _(a)-_(o′)A_(es)T_(es)T_(es) ^(m)C_(es)A_(es) ^(m)C_(es)T_(es)T_(es)T_(es) ^(m)C_(es)A_(es)T_(es)A_(es)A_(es) GalNAc₃-7a PO 4901 T_(es)G_(es) ^(m)C_(es)T_(es)G_(es)G_(e) 699821 GalNAc ₃ -7 _(a)-_(o′)A_(es)T_(eo)T_(eo) ^(m)C_(eo)A_(eo) ^(m)C_(eo)T_(eo)T_(eo)T_(eo) ^(m)C_(eo)A_(eo)T_(eo)A_(eo) GalNAc₃-7a PO 4901 A_(eo)T_(eo)G_(eo) ^(m)C_(eo)T_(es)G_(es)G_(e) 700000 A_(es)T_(es)T_(es) ^(m)C_(es)A_(es) ^(m)C_(es)T_(es)T_(es)T_(es) ^(m)C_(es)A_(es)T_(es)A_(es)A_(es)T_(es)G_(es) ^(m)C_(es)T_(es)G_(es) GalNAc₃-1a A_(d) 4902 G_(eo) A _(do′) -GalNAc ₃ -1 _(a) 703421 X-ATT^(m)CA^(m)CTTT^(m)CATAATG^(m)CTGG n/a n/a 4901 703422 GalNAc ₃ -7 _(b)-X-ATT^(m)CA^(m)CTTT^(m)CATAATG^(m)CTGG GalNAc₃-7b n/a 4901 The structure of GalNAc₃-7_(a) was shown previously in Example 48. “X” indicates a 5′ primary amine generated by Gene Tools (Philomath, Oreg.), and GalNAc₃-7_(b) indicates the structure of GalNAc₃-7_(a) lacking the —NH—C₆—O portion of the linker as shown below:

ISIS numbers 703421 and 703422 are morphlino oligonucleotides, wherein each nucleotide of the two oligonucleotides is a morpholino nucleotide. Treatment

Six week old transgenic mice that express human SMN were injected subcutaneously once with an oligonucleotide listed in Table 91 or with saline. Each treatment group consisted of 2 males and 2 females. The mice were sacrificed 3 days following the dose to determine the liver human SMN mRNA levels both with and without exon 7 using real-time PCR according to standard protocols. Total RNA was measured using Ribogreen reagent. The SMN mRNA levels were normalized to total mRNA, and further normalized to the averages for the saline treatment group. The resulting average ratios of SMN mRNA including exon 7 to SMN mRNA missing exon 7 are shown in Table 91. The results show that fully modified oligonucleotides that modulate splicing and comprise a GalNAc conjugate are significantly more potent in altering splicing in the liver than the parent oligonucleotides lacking a GlaNAc conjugate. Furthermore, this trend is maintained for multiple modification chemistries, including 2′-MOE and morpholino modified oligonucleotides.

TABLE 91 Effect of oligonucleotides targeting human SMN in vivo Dose GalNAc₃ SEQ ISIS No. (mg/kg) +Exon 7/−Exon 7 Cluster CM ID No. Saline n/a 1.00 n/a n/a n/a 387954 32 1.65 n/a n/a 4901 387954 288 5.00 n/a n/a 4901 699819 32 7.84 GalNAc₃-7a PO 4901 699821 32 7.22 GalNAc₃-7a PO 4901 700000 32 6.91 GalNAc₃-1a A_(d) 4902 703421 32 1.27 n/a n/a 4901 703422 32 4.12 GalNAc₃-7b n/a 4901

Example 89: Antisense Inhibition In Vivo by Oligonucleotides Targeting Apolipoprotein A (Apo(a)) Comprising a GalNAc₃ Conjugate

The oligonucleotides listed in Table 92 below were tested in a study for dose-dependent inhibition of Apo(a) in transgenic mice.

TABLE 92 Modified ASOs targeting Apo(a) ISIS GalNAc₃ SEQ ID No. Sequences (5′ to 3′) Cluster CM No. 494372 T_(es)G_(es) ^(m)C_(es)T_(es) ^(m)C_(es) ^(m)C_(ds) n/a n/a 4903 G_(ds)T_(ds)T_(ds)G_(ds)G_(ds)T_(ds) G_(ds) ^(m)C_(ds)T_(ds)T_(es)G_(es)T_(es) T_(es) ^(m)C_(e) 681257 GalNAc ₃ -7 _(a)-_(o′)T_(es)G_(eo) GalNAc₃-7a PO 4903 ^(m)C_(eo)T_(eo) ^(m)C_(eo) ^(m)C_(ds)G_(ds)T_(ds) T_(ds)G_(ds)G_(ds)T_(ds)G_(ds) ^(m)C_(ds) T_(ds)T_(eo)G_(eo)T_(es)T_(es) ^(m)C_(e) The structure of GalNAc₃-7a was shown in Example 48. Treatment

Eight week old, female C57BL/6 mice (Jackson Laboratory, Bar Harbor, Me.) were each injected subcutaneously once per week at a dosage shown below, for a total of six doses, with an oligonucleotide listed in Table 92 or with PBS. Each treatment group consisted of 3-4 animals. Tail bleeds were performed the day before the first dose and weekly following each dose to determine plasma Apo(a) protein levels. The mice were sacrificed two days following the final administration. Apo(a) liver mRNA levels were determined using real-time PCR and RIBOGREEN RNA quantification reagent (Molecular Probes, Inc. Eugene, Oreg.) according to standard protocols. Apo(a) plasma protein levels were determined using ELISA, and liver transaminase levels were determined. The mRNA and plasma protein results in Table 93 are presented as the treatment group average percent relative to the PBS treated group. Plasma protein levels were further normalized to the baseline (BL) value for the PBS group. Average absolute transaminase levels and body weights (% relative to baseline averages) are reported in Table 94.

As illustrated in Table 93, treatment with the oligonucleotides lowered Apo(a) liver mRNA and plasma protein levels in a dose-dependent manner. Furthermore, the oligonucleotide comprising the GalNAc conjugate was significantly more potent with a longer duration of action than the parent oligonucleotide lacking a GalNAc conjugate. As illustrated in Table 94, transaminase levels and body weights were unaffected by the oligonucleotides, indicating that the oligonucleotides were well tolerated.

TABLE 93 Apo(a) liver mRNA and plasma protein levels ISIS Dosage Apo(a) mRNA Apo(a) plasma protein (% PBS) No. (mg/kg) (% PBS) BL Week 1 Week 2 Week 3 Week 4 Week 5 Week 6 PBS n/a 100 100 120 119 113 88 121 97 494372 3 80 84 89 91 98 87 87 79 10 30 87 72 76 71 57 59 46 30 5 92 54 28 10 7 9 7 681257 0.3 75 79 76 89 98 71 94 78 1 19 79 88 66 60 54 32 24 3 2 82 52 17 7 4 6 5 10 2 79 17 6 3 2 4 5

TABLE 94 Dosage ALT AST Body weight ISIS No. (mg/kg) (U/L) (U/L) (% baseline) PBS n/a 37 54 103 494372 3 28 68 106 10 22 55 102 30 19 48 103 681257 0.3 30 80 104 1 26 47 105 3 29 62 102 10 21 52 107

Example 90: Antisense Inhibition In Vivo by Oligonucleotides Targeting TTR Comprising a GalNAc₃ Cluster

Oligonucleotides listed in Table 95 below were tested in a dose-dependent study for antisense inhibition of human transthyretin (TTR) in transgenic mice that express the human TTR gene.

Treatment

TTR transgenic mice were each injected subcutaneously once per week for three weeks, for a total of three doses, with an oligonucleotide and dosage listed in Table 96 or with PBS. Each treatment group consisted of 4 animals. Prior to the first dose, a tail bleed was performed to determine plasma TTR protein levels at baseline (BL). The mice were sacrificed 72 hours following the final administration. TTR protein levels were measured using a clinical analyzer (AU480, Beckman Coulter, CA). Real-time PCR and RIBOGREEN RNA quantification reagent (Molecular Probes, Inc. Eugene, Oreg.) were used according to standard protocols to determine liver human TTR mRNA levels. The results presented in Table 96 are the average values for each treatment group. The mRNA levels are the average values relative to the average for the PBS group. Plasma protein levels are the average values relative to the average value for the PBS group at baseline. “BL” indicates baseline, measurements that were taken just prior to the first dose. As illustrated in Table 96, treatment with antisense oligonucleotides lowered TTR expression levels in a dose-dependent manner. The oligonucleotides comprising a GalNAc conjugate were more potent than the parent lacking a GalNAc conjugate (ISIS 420915), and oligonucleotides comprising a phosphodiester or deoxyadenosine cleavable moiety showed significant improvements in potency compared to the parent lacking a conjugate (see ISIS numbers 682883 and 666943 vs 420915 and see Examples 86 and 87).

TABLE 95 Oligonucleotides targeting human TTR GalNAc SEQ Isis No. Sequence 5′ to 3′ Linkages cluster CM ID No. 420915 T_(es) ^(m)C_(es)T_(es)T_(es)G_(es)G_(ds)T_(ds)T_(ds)A_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds)A_(ds) PS n/a n/a 4899 A_(es)T_(es) ^(m)C_(es) ^(m)C_(es) ^(m)C_(e) 682883 GalNAc ₃ -3 _(a-o′)T_(es) ^(m)C_(eo)T_(eo)T_(eo)G_(eo)G_(ds)T_(ds)T_(ds)A_(ds) ^(m)C_(ds)A_(ds) PS/PO GalNAc₃-3a PO 4899 T_(ds)G_(ds)A_(ds)A_(ds)A_(eo)T_(eo) ^(m)C_(es) ^(m)C_(es) ^(m)C_(e) 666943 GalNAc ₃ -3 _(a-o′) A _(do)T_(es) ^(m)C_(eo)T_(eo)T_(eo)G_(eo)G_(ds)T_(ds)T_(ds)A_(ds) PS/PO GalNAc₃-3a A_(d) 4904 ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds)A_(ds)A_(eo)T_(eo) ^(m)C_(es) ^(m)C_(es) ^(m)C_(e) 682887 GalNAc ₃ -7 _(a-o′) A _(do)T_(es) ^(m)C_(eo)T_(eo)T_(eo)G_(eo)G_(ds)T_(ds)T_(ds)A_(ds) PS/PO GalNAc₃-7a A_(d) 4904 ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds)A_(ds)A_(eo)T_(eo) ^(m)C_(es) ^(m)C_(es) ^(m)C_(e) 682888 GalNAc ₃ -10 _(a-o′) A _(do)T_(es) ^(m)C_(eo)T_(eo)T_(eo)G_(eo)G_(ds)T_(ds)T_(ds)A_(ds) PS/PO GalNAc₃-10a A_(d) 4904 ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds)A_(ds)A_(eo)T_(eo) ^(m)C_(es) ^(m)C_(es) ^(m)C_(e) 682889 GalNAc ₃ -13 _(a-o′) A _(do)T_(es) ^(m)C_(eo)T_(eo)T_(eo)G_(eo)G_(ds)T_(ds)T_(ds)A_(ds) PS/PO GalNAc₃-13a A_(d) 4904 ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds)A_(ds)A_(eo)T_(eo) ^(m)C_(es) ^(m)C_(es) ^(m)C_(e) The legend for Table 95 can be found in Example 74. The structure of GalNAc₃-3_(a) was shown in Example 39. The structure of GalNAc₃-7_(a) was shown in Example 48. The structure of GalNAc₃-10_(a) was shown in Example 46. The structure of GalNAc₃-13_(a) was shown in Example 62.

TABLE 96 Antisense inhibition of human TTR in vivo Dosage TTR mRNA TTR protein GalNAc Isis No. (mg/kg) (% PBS) (% BL) cluster CM PBS n/a 100 124 n/a n/a 420915 6 69 114 n/a n/a 20 71 86 60 21 36 682883 0.6 61 73 GalNAc₃-3a PO 2 23 36 6 18 23 666943 0.6 74 93 GalNAc₃-3a A_(d) 2 33 57 6 17 22 682887 0.6 60 97 GalNAc₃-7a A_(d) 2 36 49 6 12 19 682888 0.6 65 92 GalNAc₃-10a A_(d) 2 32 46 6 17 22 682889 0.6 72 74 GalNAc₃-13a A_(d) 2 38 45 6 16 18

Example 91: Antisense Inhibition In Vivo by Oligonucleotides Targeting Factor VII Comprising a GalNAc₃ Conjugate in Non-Human Primates

Oligonucleotides listed in Table 97 below were tested in a non-terminal, dose escalation study for antisense inhibition of Factor VII in monkeys.

Treatment

Non-naïve monkeys were each injected subcutaneously on days 0, 15, and 29 with escalating doses of an oligonucleotide listed in Table 97 or with PBS. Each treatment group consisted of 4 males and 1 female. Prior to the first dose and at various time points thereafter, blood draws were performed to determine plasma Factor VII protein levels. Factor VII protein levels were measured by ELISA. The results presented in Table 98 are the average values for each treatment group relative to the average value for the PBS group at baseline (BL), the measurements taken just prior to the first dose. As illustrated in Table 98, treatment with antisense oligonucleotides lowered Factor VII expression levels in a dose-dependent manner, and the oligonucleotide comprising the GalNAc conjugate was significantly more potent in monkeys compared to the oligonucleotide lacking a GalNAc conjugate.

TABLE 97 Oligonucleotides targeting Factor VII GalNAc SEQ Isis No. Sequence 5′ to 3′ Linkages cluster CM ID No. 407935 A_(es)T_(es)G_(es) ^(m)C_(es)A_(es)T_(ds)G_(ds)G_(ds)T_(ds)G_(ds)A_(ds)T_(ds)G_(ds) ^(m)C_(ds)T_(ds) PS n/a n/a 4905 T_(es) ^(m)C_(es)T_(es)G_(es)A_(e) 686892 GalNAc ₃ -10 _(a-o′)A_(es)T_(es)G_(es) ^(m)C_(es)A_(es)T_(ds)G_(ds)G_(ds)T_(ds)G_(ds) PS GalNAc₃-10a PO 4905 A_(ds)T_(ds)G_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es)T_(es)G_(es)A_(e) The legend for Table 97 can be found in Example 74. The structure of GalNAc₃-10_(a) was shown in Example 46.

TABLE 98 Factor VII plasma protein levels Dose Factor VII ISIS No. Day (mg/kg) (% BL) 407935 0 n/a 100 15 10 87 22 n/a 92 29 30 77 36 n/a 46 43 n/a 43 686892 0  3 100 15 10 56 22 n/a 29 29 30 19 36 n/a 15 43 n/a 11

Example 92: Antisense Inhibition in Primary Hepatocytes by Antisense Oligonucleotides Targeting ApoCIII Comprising a GalNAc₃ Conjugate

Primary mouse hepatocytes were seeded in 96-well plates at 15,000 cells per well, and the oligonucleotides listed in Table 99, targeting mouse ApoC-III, were added at 0.46, 1.37, 4.12, or 12.35, 37.04, 111.11, or 333.33 nM or 1.00 μM. After incubation with the oligonucleotides for 24 hours, the cells were lysed and total RNA was purified using RNeasy (Qiagen). ApoC-III mRNA levels were determined using real-time PCR and RIBOGREEN® RNA quantification reagent (Molecular Probes, Inc.) according to standard protocols. IC₅₀ values were determined using Prism 4 software (GraphPad). The results show that regardless of whether the cleavable moiety was a phosphodiester or a phosphodiester-linked deoxyadensoine, the oligonucleotides comprising a GalNAc conjugate were significantly more potent than the parent oligonucleotide lacking a conjugate.

TABLE 99 Inhibition of mouse APOC-III expression in mouse primary hepatocytes ISIS IC₅₀ SEQ No. Sequence (5′ to 3′) CM (nM) ID No. 440670 ^(m)C_(es)A_(es)G_(es) ^(m)C_(es)T_(es)T_(ds)T_(ds)A_(ds)T_(ds)T_(ds)A_(ds)G_(ds)G_(ds)G_(ds)A_(ds) ^(m)C_(es)A_(es)G_(es) ^(m)C_(es)A_(e) n/a 13.20 4906 661180 ^(m)C_(es)A_(es)G_(es) ^(m)C_(es)T_(es)T_(ds)T_(ds)A_(ds)T_(ds)T_(ds)A_(ds)G_(ds)G_(ds)G_(ds)A_(ds) ^(m)C_(es) A_(d) 1.40 4907 A_(es)G_(es) ^(m)C_(es)A_(eo) A _(do′) -GalNAc ₃ -1 _(a) 680771 GalNAc ₃ -3 _(a-o′) ^(m)C_(es)A_(es)G_(es) ^(m)C_(es)T_(es)T_(ds)T_(ds)A_(ds)T_(ds)T_(ds)A_(ds)G_(ds)G_(ds)G_(ds)A_(ds) ^(m)C_(es) PO 0.70 4906 A_(es)G_(es) ^(m)C_(es)A_(e) 680772 GalNAc ₃ -7 _(a-o′) ^(m)C_(es)A_(es)G_(es) ^(m)C_(es)T_(es)T_(ds)T_(ds)A_(ds)T_(ds)T_(ds)A_(ds)G_(ds)G_(ds)G_(ds)A_(ds) ^(m)C_(es) PO 1.70 4906 A_(es)G_(es) ^(m)C_(es)A_(e) 680773 GalNAc ₃ -10 _(a-o′) ^(m)C_(es)A_(es)G_(es) ^(m)C_(es)T_(es)T_(ds)T_(ds)A_(ds)T_(ds)T_(ds)A_(ds)G_(ds)G_(ds)G_(ds)A_(ds) ^(m)C_(es) PO 2.00 4906 A_(es)G_(es) ^(m)C_(es)A_(e) 680774 GalNAc ₃ -13 _(a-o′) ^(m)C_(es)A_(es)G_(es) ^(m)C_(es)T_(es)T_(ds)T_(ds)A_(ds)T_(ds)T_(ds)A_(ds)G_(ds)G_(ds)G_(ds)A_(ds) ^(m)C_(es) PO 1.50 4906 A_(es)G_(es) ^(m)C_(es)A_(e) 681272 GalNAc ₃ -3 _(a-o′) ^(m)C_(es)A_(eo)G_(eo) ^(m)C_(eo)T_(eo)T_(ds)T_(ds)A_(ds)T_(ds)T_(ds)A_(ds)G_(ds)G_(ds)G_(ds)A_(ds) ^(m)C_(eo) PO <0.46 4906 A_(eo)G_(es) ^(m)C_(es)A_(e) 681273 GalNAc ₃ -3 _(a)-_(o′) A _(do) ^(m)C_(es)A_(es)G_(es) ^(m)C_(es)T_(es)T_(ds)T_(ds)A_(ds)T_(ds)T_(ds)A_(ds)G_(ds)G_(ds)G_(ds)A_(ds) A_(d) 1.10 4908 ^(m)C_(es)A_(es)G_(es) ^(m)C_(es)A_(e) 683733 ^(m)C_(es)A_(es)G_(es) ^(m)C_(es)T_(es)T_(ds)T_(ds)A_(ds)T_(ds)T_(ds)A_(ds)G_(ds)G_(ds)G_(ds)A_(ds) ^(m)C_(es) A_(d) 2.50 4907 A_(es)G_(es) ^(m)C_(es)A_(eo) A _(do′) -GalNAc ₃ -19 _(a) The structure of GalNAc₃-1_(a) was shown previously in Example 9, GalNAc₃-3_(a) was shown in Example 39, GalNAc₃-7_(a) was shown in Example 48, GalNAc₃-10_(a) was shown in Example 46, GalNAc₃-13_(a) was shown in Example 62, and GalNAc₃-19_(a) was shown in Example 70.

Example 93: Antisense Inhibition In Vivo by Oligonucleotides Targeting SRB-1 Comprising Mixed Wings and a 5′-GalNAc₃ Conjugate

The oligonucleotides listed in Table 100 were tested in a dose-dependent study for antisense inhibition of SRB-1 in mice.

TABLE 100 Modified ASOs targeting SRB-1 ISIS GalNAc₃ SEQ No. Sequences (5′ to 3′) Cluster CM ID No. 449093 T_(ks)T_(ks) ^(m)C_(ks)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(ks) ^(m)C_(ks) ^(m)C_(k) n/a n/a 4909 699806 GalNAc ₃ -3 _(a)-_(o′)T_(ks)T_(ks) ^(m)C_(ks)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds) GalNAc₃-3a PO 4909 T_(ds)T_(ks) ^(m)C_(ks) ^(m)C_(k) 699807 GalNAc ₃ -7 _(a)-_(o′)T_(ks)T_(ks) ^(m)C_(ks)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds) GalNAc₃-7a PO 4909 T_(ds)T_(ks) ^(m)C_(ks) ^(m)C_(k) 699809 GalNAc ₃ -7 _(a)-_(o′)T_(ks)T_(ks) ^(m)C_(ks)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds) GalNAc₃-7a PO 4909 T_(ds)T_(es) ^(m)C_(es) ^(m)C_(e) 699811 GalNAc ₃ -7 _(a)-_(o′)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds) GalNAc₃-7a PO 4909 T_(ds)T_(ks) ^(m)C_(ks) ^(m)C_(k) 699813 GalNAc ₃ -7 _(a)-_(o′)T_(ks)T_(ds) ^(m)C_(ks)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds) GalNAc₃-7a PO 4909 T_(ds)T_(ks) ^(m)C_(ds) ^(m)C_(k) 699815 GalNAc ₃ -7 _(a)-_(o′)T_(es)T_(ks) ^(m)C_(ks)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds) GalNAc₃-7a PO 4909 T_(ds)T_(ks) ^(m)C_(ks) ^(m)C_(e)

The structure of GalNAc₃-3_(a) was shown previously in Example 39, and the structure of GalNAc₃-7a was shown previously in Example 48. Subscripts: “e” indicates 2′-MOE modified nucleoside; “d” indicates β-D-2′-deoxyribonucleoside; “k” indicates 6′-(S)—CH₃ bicyclic nucleoside (cEt); “s” indicates phosphorothioate internucleoside linkages (PS); “o” indicates phosphodiester internucleoside linkages (PO). Superscript “m” indicates 5-methylcytosines.

Treatment

Six to eight week old C57BL/6 mice (Jackson Laboratory, Bar Harbor, Me.) were injected subcutaneously once at the dosage shown below with an oligonucleotide listed in Table 100 or with saline. Each treatment group consisted of 4 animals. The mice were sacrificed 72 hours following the final administration. Liver SRB-1 mRNA levels were measured using real-time PCR. SRB-1 mRNA levels were normalized to cyclophilin mRNA levels according to standard protocols. The results are presented as the average percent of SRB-1 mRNA levels for each treatment group relative to the saline control group. As illustrated in Table 101, treatment with antisense oligonucleotides lowered SRB-1 mRNA levels in a dose-dependent manner, and the gapmer oligonucleotides comprising a GalNAc conjugate and having wings that were either full cEt or mixed sugar modifications were significantly more potent than the parent oligonucleotide lacking a conjugate and comprising full cEt modified wings.

Body weights, liver transaminases, total bilirubin, and BUN were also measured, and the average values for each treatment group are shown in Table 101. Body weight is shown as the average percent body weight relative to the baseline body weight (% BL) measured just prior to the oligonucleotide dose.

TABLE 101 SRB-1 mRNA, ALT, AST, BUN, and total bilirubin levels and body weights Body ISIS Dosage SRB-1 mRNA ALT AST weight No. (mg/kg) (% PBS) (U/L) (U/L) Bil BUN (% BL) PBS n/a 100 31 84 0.15 28 102 449093 1 111 18 48 0.17 31 104 3 94 20 43 0.15 26 103 10 36 19 50 0.12 29 104 699806 0.1 114 23 58 0.13 26 107 0.3 59 21 45 0.12 27 108 1 25 30 61 0.12 30 104 699807 0.1 121 19 41 0.14 25 100 0.3 73 23 56 0.13 26 105 1 24 22 69 0.14 25 102 699809 0.1 125 23 57 0.14 26 104 0.3 70 20 49 0.10 25 105 1 33 34 62 0.17 25 107 699811 0.1 123 48 77 0.14 24 106 0.3 94 20 45 0.13 25 101 1 66 57 104 0.14 24 107 699813 0.1 95 20 58 0.13 28 104 0.3 98 22 61 0.17 28 105 1 49 19 47 0.11 27 106 699815 0.1 93 30 79 0.17 25 105 0.3 64 30 61 0.12 26 105 1 24 18 41 0.14 25 106

Example 94: Antisense Inhibition In Vivo by Oligonucleotides Targeting SRB-1 Comprising 2′-Sugar Modifications and a 5′-GalNAc₃ Conjugate

The oligonucleotides listed in Table 102 were tested in a dose-dependent study for antisense inhibition of SRB-1 in mice.

TABLE 102 Modified ASOs targeting SRB-1 ISIS GalNAc₃ SEQ No. Sequences (5′ to 3′) Cluster CM ID No. 353382 G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es) n/a n/a 4886 T_(es)T_(e) 700989 G_(ms)C_(ms)U_(ms)U_(ms)C_(ms)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)U_(ms)C_(ms)C_(ms) n/a n/a 4910 U_(ms)U_(m) 666904 GalNAc ₃ -3 _(a) - _(o′)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) GalNAc₃-3a PO 4886 ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) 700991 GalNAc ₃ -7 _(a) - _(o′)G_(ms)C_(ms)U_(ms)U_(ms)C_(ms)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds) GalNAc₃-7a PO 4910 A_(ds) ^(m)C_(ds)T_(ds)U_(ms)C_(ms)C_(ms)U_(ms)U_(m) Subscript “m” indicates a 2′-O-methyl modified nucleoside. See Example 74 for complete table legend. The structure of GalNAc₃-3_(a) was shown previously in Example 39, and the structure of GalNAc₃-7a was shown previously in Example 48. Treatment

The study was completed using the protocol described in Example 93. Results are shown in Table 103 below and show that both the 2′-MOE and 2′-OMe modified oligonucleotides comprising a GalNAc conjugate were significantly more potent than the respective parent oligonucleotides lacking a conjugate. The results of the body weights, liver transaminases, total bilirubin, and BUN measurements indicated that the compounds were all well tolerated.

TABLE 103 SRB-1 mRNA Dosage SRB-1 mRNA ISIS No. (mg/kg) (% PBS) PBS n/a 100 353382 5 116 15 58 45 27 700989 5 120 15 92 45 46 666904 1 98 3 45 10 17 700991 1 118 3 63 10 14

Example 95: Antisense Inhibition In Vivo by Oligonucleotides Targeting SRB-1 Comprising Bicyclic Nucleosides and a 5′-GalNAc₃ Conjugate

The oligonucleotides listed in Table 104 were tested in a dose-dependent study for antisense inhibition of SRB-1 in mice.

TABLE 104 Modified ASOs targeting SRB-1 ISIS GalNAc₃ SEQ No. Sequences (5′ to 3′) Cluster CM ID No 440762 T_(ks) ^(m)C_(ks)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(ks) ^(m)C_(k) n/a n/a 4880 666905 GalNAc ₃ -3 _(a) - _(o′)T_(ks) ^(m)C_(ks)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds) A_(ds) ^(m)C_(ds)T_(ds)T_(ks) ^(m)C_(k) GalNAc₃-3_(a) PO 4880 699782 GalNAc ₃ -7 _(a) - _(o′)T_(ks) ^(m)C_(ks)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(ks) ^(m)C_(k) GalNAc₃-7_(a) PO 4880 699783 GalNAc ₃ -3 _(a) - _(o′)T_(ls) ^(m)C_(ls)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(ls) ^(m)C_(l) GalNAc₃-3_(a) PO 4880 653621 T_(ls) ^(m)C_(ls)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(ls) ^(m)C_(lo) A _(do′) -GalNAc ₃ -1 _(a) GalNAc₃-1_(a) A_(d) 4881 439879 T_(gs) ^(m)C_(gs)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(d)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(gs) ^(m)C_(g) n/a n/a 4880 699789 GalNAc ₃ -3 _(a) - _(o′)T_(gs) ^(m)C_(gs)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(d)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(gs) ^(m)C_(g) GalNAc₃-3_(a) PO 4880 Subscript “g” indicates a fluoro-HNA nucleoside, subscript “1” indicates a locked nucleoside comprising a 2′-O—CH₂-4′ bridge. See the Example 74 table legend for other abbreviations. The structure of GalNAc₃-1_(a) was shown previously in Example 9, the structure of GalNAc₃-3_(a) was shown previously in Example 39, and the structure of GalNAc₃-7a was shown previously in Example 48. Treatment

The study was completed using the protocol described in Example 93. Results are shown in Table 105 below and show that oligonucleotides comprising a GalNAc conjugate and various bicyclic nucleoside modifications were significantly more potent than the parent oligonucleotide lacking a conjugate and comprising bicyclic nucleoside modifications. Furthermore, the oligonucleotide comprising a GalNAc conjugate and fluoro-HNA modifications was significantly more potent than the parent lacking a conjugate and comprising fluoro-HNA modifications. The results of the body weights, liver transaminases, total bilirubin, and BUN measurements indicated that the compounds were all well tolerated.

TABLE 105 SRB-1 mRNA, ALT, AST, BUN, and total bilirubin levels and body weights Dosage SRB-1 mRNA ISIS No. (mg/kg) (% PBS) PBS n/a 100 440762 1 104 3 65 10 35 666905 0.1 105 0.3 56 1 18 699782 0.1 93 0.3 63 1 15 699783 0.1 105 0.3 53 1 12 653621 0.1 109 0.3 82 1 27 439879 1 96 3 77 10 37 699789 0.1 82 0.3 69 1 26

Example 96: Plasma Protein Binding of Antisense Oligonucleotides Comprising a GalNAc₃ Conjugate Group

Oligonucleotides listed in Table 70 targeting ApoC-III and oligonucleotides in Table 106 targeting Apo(a) were tested in an ultra-filtration assay in order to assess plasma protein binding.

TABLE 106 Modified oligonucleotides targeting Apo(a) ISIS GalNAc₃ SEQ No. Sequences (5′ to 3′) Cluster CM ID No 494372 T_(es)G_(es) ^(m)C_(es)T_(es) ^(m)C_(es) ^(m)C_(ds)G_(ds)T_(ds)T_(ds)G_(ds)G_(ds)T_(ds)G_(ds) ^(m)C_(ds)T_(ds)T_(es)G_(es)T_(es) n/a n/a 4903 T_(es) ^(m)C_(e) 693401 T_(es)G_(eo) ^(m)C_(eo)T_(eo) ^(m)C_(eo) ^(m)C_(ds)G_(ds)T_(ds)T_(ds)G_(ds)G_(ds)T_(ds)G_(ds) ^(m)C_(ds)T_(ds)T_(eo)G_(eo)T_(es) n/a n/a 4903 T_(es) ^(m)C_(e) 681251 GalNAc ₃ -7 _(a) - _(o′)T_(es)G_(es) ^(m)C_(es)T_(es) ^(m)C_(es) ^(m)C_(ds)G_(ds)T_(ds)T_(ds)G_(ds)G_(ds)T_(ds)G_(ds) ^(m)C_(ds) GalNAc₃-7_(a) PO 4903 T_(ds)T_(es)G_(es)T_(es)T_(es) ^(m)C_(e) 681257 GalNAc ₃ -7 _(a) - _(o′)T_(es)G_(eo) ^(m)C_(eo)T_(eo) ^(m)C_(eo) ^(m)C_(ds)G_(ds)T_(ds)T_(ds)G_(ds)G_(ds)T_(ds)G_(ds) ^(m)C_(ds) GalNAc₃-7_(a) PO 4903 T_(ds)T_(eo)G_(eo)T_(es)T_(es) ^(m)C_(e) See the Example 74 for table legend. The structure of GalNAc₃-7a was shown previously in Example 48.

Ultrafree-MC ultrafiltration units (30,000 NMWL, low-binding regenerated cellulose membrane, Millipore, Bedford, Mass.) were pre-conditioned with 300 μL of 0.5% Tween 80 and centrifuged at 2000 g for 10 minutes, then with 300 μL of a 300 μg/mL solution of a control oligonucleotide in H₂O and centrifuged at 2000 g for 16 minutes. In order to assess non-specific binding to the filters of each test oligonucleotide from Tables 70 and 106 to be used in the studies, 300 μL of a 250 ng/mL solution of oligonucleotide in H₂O at pH 7.4 was placed in the pre-conditioned filters and centrifuged at 2000 g for 16 minutes. The unfiltered and filtered samples were analyzed by an ELISA assay to determine the oligonucleotide concentrations. Three replicates were used to obtain an average concentration for each sample. The average concentration of the filtered sample relative to the unfiltered sample is used to determine the percent of oligonucleotide that is recovered through the filter in the absence of plasma (% recovery).

Frozen whole plasma samples collected in K3-EDTA from normal, drug-free human volunteers, cynomolgus monkeys, and CD-1 mice, were purchased from Bioreclamation LLC (Westbury, N.Y.). The test oligonucleotides were added to 1.2 mL aliquots of plasma at two concentrations (5 and 150 μg/mL). An aliquot (300 μL) of each spiked plasma sample was placed in a pre-conditioned filter unit and incubated at 37° C. for 30 minutes, immediately followed by centrifugation at 2000 g for 16 minutes. Aliquots of filtered and unfiltered spiked plasma samples were analyzed by an ELISA to determine the oligonucleotide concentration in each sample. Three replicates per concentration were used to determine the average percentage of bound and unbound oligonucleotide in each sample. The average concentration of the filtered sample relative to the concentration of the unfiltered sample is used to determine the percent of oligonucleotide in the plasma that is not bound to plasma proteins (% unbound). The final unbound oligonucleotide values are corrected for non-specific binding by dividing the % unbound by the % recovery for each oligonucleotide. The final % bound oligonucleotide values are determined by subtracting the final % unbound values from 100. The results are shown in Table 107 for the two concentrations of oligonucleotide tested (5 and 150 μg/mL) in each species of plasma. The results show that GalNAc conjugate groups do not have a significant impact on plasma protein binding. Furthermore, oligonucleotides with full PS internucleoside linkages and mixed PO/PS linkages both bind plasma proteins, and those with full PS linkages bind plasma proteins to a somewhat greater extent than those with mixed PO/PS linkages.

TABLE 107 Percent of modified oligonucleotide bound to plasma proteins ISIS Human plasma Monkey plasma Mouse plasma No. 5 μg/mL 150 μg/mL 5 μg/mL 150 μg/mL 5 μg/mL 150 μg/mL 304801 99.2 98.0 99.8 99.5 98.1 97.2 663083 97.8 90.9 99.3 99.3 96.5 93.0 674450 96.2 97.0 98.6 94.4 94.6 89.3 494372 94.1 89.3 98.9 97.5 97.2 93.6 693401 93.6 89.9 96.7 92.0 94.6 90.2 681251 95.4 93.9 99.1 98.2 97.8 96.1 681257 93.4 90.5 97.6 93.7 95.6 92.7

Example 97: Modified Oligonucleotides Targeting TTR Comprising a GalNAc₃ Conjugate Group

The oligonucleotides shown in Table 108 comprising a GalNAc conjugate were designed to target TTR.

TABLE 108 Modified oligonucleotides targeting TTR GalNAc₃ SEQ ID ISIS No. Sequences (5′ to 3′) Cluster CM No 666941 GalNAc ₃ -3 _(a) - _(o′) A _(do) T_(es) ^(m) C_(es) T_(es) T_(es) G_(es) G_(ds) T_(ds) T_(ds) A_(ds) ^(m)C_(ds) GalNAc₃-3 A_(d) 4904 A_(ds) T_(ds) G_(ds) A_(ds) A_(ds) A_(es) T_(es) ^(m)C_(es) ^(m)C_(es) ^(m)C_(e) 666942 T_(es) ^(m) C_(eo) T_(eo) T_(eo) G_(eo) G_(ds) T_(ds) T_(ds) A_(ds) ^(m)C_(ds) A_(ds) T_(ds) G_(ds) A_(ds) A_(ds) GalNAc₃-1 A_(d) 4904 A_(eo) T_(eo) ^(m)C_(es) ^(m)C_(es) ^(m)C_(eo) A _(do′) -GalNAc ₃ -3 _(a) 682876 GalNAc ₃ -3 _(a) - _(o′)T_(es) ^(m)C_(es) T_(es) T_(es) G_(es) G_(ds) T_(ds) T_(ds) A_(ds) ^(m)C_(ds) A_(ds) T_(ds) GalNAc₃-3 PO 4899 G_(ds) A_(ds) A_(ds) A_(es) T_(es) ^(m)C_(es) ^(m)C_(es) ^(m)C_(e) 682877 GalNAc ₃ -7 _(a) - _(o′)T_(es) ^(m) C_(es) T_(es) T_(es) G_(es) G_(ds) T_(ds) T_(ds) A_(ds) ^(m)C_(ds) A_(ds) T_(ds) GalNAc₃-7 PO 4899 G_(ds) A_(ds) A_(ds) A_(es) T_(es) ^(m)C_(es) ^(m)C_(es) ^(m)C_(e) 682878 GalNAc ₃ -10 _(a) - _(o′)T_(es) ^(m)C_(es) T_(es) T_(es) G_(es) G_(ds) T_(ds) T_(ds) A_(ds) ^(m)C_(ds) A_(ds) GalNAc₃-10 PO 4899 T_(ds) G_(ds) A_(ds) A_(ds) A_(es) T_(es) ^(m)C_(es) ^(m)C_(es) ^(m)C_(e) 682879 GalNAc ₃ -13 _(a) - _(o′)T_(es) ^(m)C_(es) T_(es) T_(es) G_(es) G_(ds) T_(ds) T_(ds) A_(ds) ^(m)C_(ds) A_(ds) GalNAc₃-13 PO 4899 T_(ds) G_(ds) A_(ds) A_(ds) A_(es) T_(es) ^(m)C_(es) ^(m)C_(es) ^(m)C_(e) 682880 GalNAc ₃ -7 _(a) - _(o′) A _(do) T_(es) ^(m)C_(es) T_(es) T_(es) G_(es) G_(ds) T_(ds) T_(ds) A_(ds) ^(m)C_(ds) GalNAc₃-7 A_(d) 4904 A_(ds) T_(ds) G_(ds) A_(ds) A_(ds) A_(es) T_(es) ^(m)C_(es) ^(m)C_(es) ^(m)C_(e) 682881 GalNAc ₃ -10 _(a) - _(o′) A _(do) T_(es) ^(m)C_(es) T_(es) T_(es) G_(es) G_(ds) T_(ds) T_(ds) A_(ds) ^(m)C_(ds) GalNAc₃-10 A_(d) 4904 A_(ds) T_(ds) G_(ds) A_(ds) A_(ds) A_(es) T_(es) ^(m)C_(es) ^(m)C_(es) ^(m)C_(e) 682882 GalNAc ₃ -13 _(a) - _(o′) A _(do) T_(es) ^(m)C_(es) T_(es) T_(es) G_(es) G_(ds) T_(ds) T_(ds) A_(ds) ^(m)C_(ds) GalNAc₃-13 A_(d) 4904 A_(ds) T_(ds) G_(ds) A_(ds) A_(ds) A_(es) T_(es) ^(m)C_(es) ^(m)C_(es) ^(m)C_(e) 684056 T_(es) ^(m) C_(es) T_(es) T_(es) G_(es) G_(ds) T_(ds) T_(ds) A_(ds) ^(m)C_(ds) A_(ds) T_(ds) G_(ds) A_(ds) A_(ds) GalNAc₃-19 A_(d) 4900 A_(es) T_(es) ^(m)C_(es) ^(m)C_(es) ^(m)C_(eo) A _(do′) -GalNAc ₃ -19 _(a) The legend for Table 108 can be found in Example 74. The structure of GalNAc₃-1 was shown in Example 9. The structure of GalNAc₃-3_(a) was shown in Example 39. The structure of GalNAc₃-7_(a) was shown in Example 48. The structure of GalNAc₃-10_(a) was shown in Example 46. The structure of GalNAc₃-13_(a) was shown in Example 62. The structure of GalNAc₃-19_(a) was shown in Example 70.

Example 98: Evaluation of Pro-Inflammatory Effects of Oligonucleotides Comprising a GalNAc Conjugate in hPMBC Assay

The oligonucleotides listed in Table 109 and were tested for pro-inflammatory effects in an hPMBC assay as described in Examples 23 and 24. (See Tables 30, 83, 95, and 108 for descriptions of the oligonucleotides.) ISIS 353512 is a high responder used as a positive control, and the other oligonucleotides are described in Tables 83, 95, and 108. The results shown in Table 109 were obtained using blood from one volunteer donor. The results show that the oligonucleotides comprising mixed PO/PS internucleoside linkages produced significantly lower pro-inflammatory responses compared to the same oligonucleotides having full PS linkages. Furthermore, the GalNAc conjugate group did not have a significant effect in this assay.

TABLE 109 GalNAc₃ ISIS No. E_(max)/EC₅₀ cluster Linkages CM 353512 3630 n/a PS n/a 420915 802 n/a PS n/a 682881 1311 GalNAc₃-10 PS A_(d) 682888 0.26 GalNAc₃-10 PO/PS A_(d) 684057 1.03 GalNAc₃-19 PO/PS A_(d)

Example 99: Binding Affinities of Oligonucleotides Comprising a GalNAc Conjugate for the Asialoglycoprotein Receptor

The binding affinities of the oligonucleotides listed in Table 110 (see Table 76 for descriptions of the oligonucleotides) for the asialoglycoprotein receptor were tested in a competitive receptor binding assay. The competitor ligand, α1-acid glycoprotein (AGP), was incubated in 50 mM sodium acetate buffer (pH 5) with 1 U neuraminidase-agarose for 16 hours at 37° C., and >90% desialylation was confirmed by either sialic acid assay or size exclusion chromatography (SEC). Iodine monochloride was used to iodinate the AGP according to the procedure by Atsma et al. (see J Lipid Res. 1991 January; 32(1): 173-81.) In this method, desialylated α1-acid glycoprotein (de-AGP) was added to 10 mM iodine chloride, Na¹²⁵I, and 1 μM glycine in 0.25 μM NaOH. After incubation for 10 minutes at room temperature, ¹²⁵I-labeled de-AGP was separated from free ¹²⁵I by concentrating the mixture twice utilizing a 3 KDMWCO spin column. The protein was tested for labeling efficiency and purity on a HPLC system equipped with an Agilent SEC-3 column (7.8×300 mm) and a ß-RAM counter. Competition experiments utilizing ¹²⁵I-labeled de-AGP and various GalNAc-cluster containing ASOs were performed as follows. Human HepG2 cells (10⁶ cells/ml) were plated on 6-well plates in 2 ml of appropriate growth media. MEM media supplemented with 10% fetal bovine serum (FBS), 2 mM L-Glutamine and 10 mM HEPES was used. Cells were incubated 16-20 hours @ 37° C. with 5% and 10% CO₂ respectively. Cells were washed with media without FBS prior to the experiment. Cells were incubated for 30 min @37° C. with 1 ml competition mix containing appropriate growth media with 2% FBS, 10⁻⁸ μM ¹²⁵I-labeled de-AGP and GalNAc-cluster containing ASOs at concentrations ranging from 10⁻¹¹ to 10⁻⁵ μM. Non-specific binding was determined in the presence of 10⁻² μM GalNAc sugar. Cells were washed twice with media without FBS to remove unbound ¹²⁵I-labeled de-AGP and competitor GalNAc ASO. Cells were lysed using Qiagen's RLT buffer containing 1% β-mercaptoethanol. Lysates were transferred to round bottom assay tubes after a brief 10 min freeze/thaw cycle and assayed on a γ-counter. Non-specific binding was subtracted before dividing ¹²⁵I protein counts by the value of the lowest GalNAc-ASO concentration counts. The inhibition curves were fitted according to a single site competition binding equation using a nonlinear regression algorithm to calculate the binding affinities (K_(D)'s).

The results in Table 110 were obtained from experiments performed on five different days. Results for oligonucleotides marked with superscript “a” are the average of experiments run on two different days. The results show that the oligonucleotides comprising a GalNAc conjugate group on the 5′-end bound the asialoglycoprotein receptor on human HepG2 cells with 1.5 to 16-fold greater affinity than the oligonucleotides comprising a GalNAc conjugate group on the 3′-end.

TABLE 110 Asialoglycoprotein receptor binding assay results Oligonucleotide end to GalNAc which GalNAc K_(D) ISIS No. conjugate conjugate is attached (nM) 661161^(a) GalNAc₃-3 5′ 3.7 666881^(a) GalNAc₃-10 5′ 7.6 666981  GalNAc₃-7 5′ 6.0 670061  GalNAc₃-13 5′ 7.4 655861^(a) GalNAc₃-1 3′ 11.6 677841^(a) GalNAc₃-19 3′ 60.8

Example 100: Antisense Inhibition In Vivo by Oligonucleotides Comprising a GalNAc Conjugate Group Targeting Apo(a) In Vivo

The oligonucleotides listed in Table 111a below were tested in a single dose study for duration of action in mice.

TABLE 111a Modified ASOs targeting APO(a) ISIS GalNAc₃ SEQ No. Sequences (5′ to 3′) Cluster CM ID No. 681251 GalNAc ₃ -7 _(a) - _(o′)T_(es)G_(es) ^(m)C_(es)T_(es) ^(m)C_(es) ^(m)C_(ds)G_(ds)T_(ds)T_(ds)G_(ds)G_(ds) GalNAc₃-7a PO 4903 T_(ds)G_(ds) ^(m)C_(ds)T_(ds)T_(es)G_(es)T_(es)T_(es) ^(m)C_(e) 681257 GalNAc ₃ -7 _(a) - _(o′)T_(es)G_(eo) ^(m)C_(eo)T_(eo) ^(m)C_(eo) ^(m)C_(ds)G_(ds)T_(ds)T_(ds)G_(ds)G_(ds) GalNAc₃-7a PO 4903 T_(ds)G_(ds) ^(m)C_(ds)T_(ds)T_(eo)G_(eo)T_(es)T_(es) ^(m)C_(e)

The structure of GalNAc₃-7_(a) was shown in Example 48.

Treatment

Female transgenic mice that express human Apo(a) were each injected subcutaneously once per week, for a total of 6 doses, with an oligonucleotide and dosage listed in Table 111b or with PBS. Each treatment group consisted of 3 animals. Blood was drawn the day before dosing to determine baseline levels of Apo(a) protein in plasma and at 72 hours, 1 week, and 2 weeks following the first dose. Additional blood draws will occur at 3 weeks, 4 weeks, 5 weeks, and 6 weeks following the first dose. Plasma Apo(a) protein levels were measured using an ELISA. The results in Table 11 lb are presented as the average percent of plasma Apo(a) protein levels for each treatment group, normalized to baseline levels (% BL), The results show that the oligonucleotides comprising a GalNAc conjugate group exhibited potent reduction in Apo(a) expression. This potent effect was observed for the oligonucleotide that comprises full PS internucleoside linkages and the oligonucleotide that comprises mixed PO and PS linkages.

TABLE 111b Apo(a) plasma protein levels Apo(a) at Apo(a) at Apo(a) at Dosage 72 hours 1 week 3 weeks ISIS No. (mg/kg) (% BL) (% BL) (% BL) PBS n/a 116 104 107 681251 0.3 97 108 93 1.0 85 77 57 3.0 54 49 11 10.0 23 15 4 681257 0.3 114 138 104 1.0 91 98 54 3.0 69 40 6 10.0 30 21 4

Example 101: Antisense Inhibition by Oligonucleotides Comprising a GalNAc Cluster Linked Via a Stable Moiety

The oligonucleotides listed in Table 112 were tested for inhibition of mouse APOC-III expression in vivo. C57Bl/6 mice were each injected subcutaneously once with an oligonucleotide listed in Table 112 or with PBS. Each treatment group consisted of 4 animals. Each mouse treated with ISIS 440670 received a dose of 2, 6, 20, or 60 mg/kg. Each mouse treated with ISIS 680772 or 696847 received 0.6, 2, 6, or 20 mg/kg. The GalNAc conjugate group of ISIS 696847 is linked via a stable moiety, a phosphorothioate linkage instead of a readily cleavable phosphodiester containing linkage. The animals were sacrificed 72 hours after the dose. Liver APOC-III mRNA levels were measured using real-time PCR. APOC-III mRNA levels were normalized to cyclophilin mRNA levels according to standard protocols. The results are presented in Table 112 as the average percent of APOC-III mRNA levels for each treatment group relative to the saline control group. The results show that the oligonucleotides comprising a GalNAc conjugate group were significantly more potent than the oligonucleotide lacking a conjugate group. Furthermore, the oligonucleotide comprising a GalNAc conjugate group linked to the oligonucleotide via a cleavable moiety (ISIS 680772) was even more potent than the oligonucleotide comprising a GalNAc conjugate group linked to the oligonucleotide via a stable moiety (ISIS 696847).

TABLE 112 Modified oligonucleotides targeting mouse APOC-III APOC-III ISIS Dosage mRNA (% SEQ No. Sequences (5′ to 3′) CM (mg/kg) PBS) ID No. 440670 ^(m)C_(es)A_(es)G_(es) ^(m)C_(es)T_(es)T_(ds)T_(ds)A_(ds)T_(ds)T_(ds)A_(ds) n/a 2 92 4906 G_(ds)G_(ds)G_(ds)A_(ds) ^(m)C_(es)A_(es)G_(es) ^(m)C_(es)A_(e) 6 86 20 59 60 37 680772 GalNAc ₃ -7 _(a-o′) ^(m)C_(es)A_(es)G_(es) ^(m)C_(es)T_(es)T_(ds)T_(ds)A_(ds) PO 0.6 79 4906 T_(ds)T_(ds)A_(ds)G_(ds)G_(ds)G_(ds)A_(ds) ^(m)C_(es)A_(es)G_(es) ^(m)C_(es)A_(e) 2 58 6 31 20 13 696847 GalNAc ₃ -7 _(a-s′) ^(m)C_(es)A_(es)G_(es) ^(m)C_(es)T_(es)T_(ds)T_(ds)A_(ds)T_(ds) n/a (PS) 0.6 83 4906 T_(ds)A_(ds)G_(ds)G_(ds)G_(ds)A_(ds) ^(m)C_(es)A_(es)G_(es) ^(m)C_(es)A_(e) 2 73 6 40 20 28

The structure of GalNAc₃-7_(a) was shown in Example 48.

Example 102: Distribution in Liver of Antisense Oligonucleotides Comprising a GalNAc Conjugate

The liver distribution of ISIS 353382 (see Table 36) that does not comprise a GalNAc conjugate and ISIS 655861 (see Table 36) that does comprise a GalNAc conjugate was evaluated. Male balb/c mice were subcutaneously injected once with ISIS 353382 or 655861 at a dosage listed in Table 113. Each treatment group consisted of 3 animals except for the 18 mg/kg group for ISIS 655861, which consisted of 2 animals. The animals were sacrificed 48 hours following the dose to determine the liver distribution of the oligonucleotides. In order to measure the number of antisense oligonucleotide molecules per cell, a Ruthenium (II) tris-bipyridine tag (MSD TAG, Meso Scale Discovery) was conjugated to an oligonucleotide probe used to detect the antisense oligonucleotides. The results presented in Table 113 are the average concentrations of oligonucleotide for each treatment group in units of millions of oligonucleotide molecules per cell. The results show that at equivalent doses, the oligonucleotide comprising a GalNAc conjugate was present at higher concentrations in the total liver and in hepatocytes than the oligonucleotide that does not comprise a GalNAc conjugate. Furthermore, the oligonucleotide comprising a GalNAc conjugate was present at lower concentrations in non-parenchymal liver cells than the oligonucleotide that does not comprise a GalNAc conjugate. And while the concentrations of ISIS 655861 in hepatocytes and non-parenchymal liver cells were similar per cell, the liver is approximately 80% hepatocytes by volume. Thus, the majority of the ISIS 655861 oligonucleotide that was present in the liver was found in hepatocytes, whereas the majority of the ISIS 353382 oligonucleotide that was present in the liver was found in non-parenchymal liver cells.

TABLE 113 Concentration in non- Concentration Concentration parenchymal in whole liver in hepatocytes liver cells Dosage (molecules* (molecules* (molecules* ISIS No. (mg/kg) 10{circumflex over ( )}6 per cell) 10{circumflex over ( )}6 per cell) 10{circumflex over ( )}6 per cell) 353382 3 9.7 1.2 37.2 10 17.3 4.5 34.0 20 23.6 6.6 65.6 30 29.1 11.7 80.0 60 73.4 14.8 98.0 90 89.6 18.5 119.9 655861 0.5 2.6 2.9 3.2 1 6.2 7.0 8.8 3 19.1 25.1 28.5 6 44.1 48.7 55.0 18 76.6 82.3 77.1

Example 103: Duration of Action In Vivo of Oligonucleotides Targeting APOC-III Comprising a GalNAc₃ Conjugate

The oligonucleotides listed in Table 114 below were tested in a single dose study for duration of action in mice.

TABLE 114 Modified ASOs targeting APOC-III ISIS GalNAc₃ SEQ No. Sequences (5′ to 3′) Cluster CM ID No. 304801 A_(es)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(ds)T_(ds)T_(ds)G_(ds)T_(ds) ^(m)C_(ds) ^(m)C_(ds)A_(ds)G_(ds) ^(m)C_(ds)T_(es)T_(es) n/a n/a 4878 T_(es)A_(es)T_(e) 663084 GalNAc ₃ -3 _(a) - _(o′) A _(do)A_(es)G_(eo) ^(m)C_(eo)T_(eo)T_(eo) ^(m)C_(ds)T_(ds)T_(ds)G_(ds)T_(ds) ^(m)C_(ds) GalNAc₃-3a A_(d) 4894 ^(m)C_(ds)A_(ds)G_(ds) ^(m)C_(ds)T_(eo)T_(eo)T_(es)A_(es)T_(e) 679241 A_(es)G_(eo) ^(m)C_(eo)T_(eo)T_(eo) ^(m)C_(ds)T_(ds)T_(ds)G_(ds)T_(ds) ^(m)C_(ds) ^(m)C_(ds)A_(ds)G_(ds) ^(m)C_(ds)T_(eo)T_(eo) GalNAc₃-19a A_(d) 4879 T_(es)A_(es)T_(eo) A _(do′) -GalNAc ₃ -19 _(a)

The structure of GalNAc₃-3_(a) was shown in Example 39, and GalNAc₃-19_(a) was shown in Example 70.

Treatment

Female transgenic mice that express human APOC-III were each injected subcutaneously once with an oligonucleotide listed in Table 114 or with PBS. Each treatment group consisted of 3 animals. Blood was drawn before dosing to determine baseline and at 3, 7, 14, 21, 28, 35, and 42 days following the dose. Plasma triglyceride and APOC-III protein levels were measured as described in Example 20. The results in Table 115 are presented as the average percent of plasma triglyceride and APOC-III levels for each treatment group, normalized to baseline levels. A comparison of the results in Table 71 of example 79 with the results in Table 115 below show that oligonucleotides comprising a mixture of phosphodiester and phosphorothioate internucleoside linkages exhibited increased duration of action than equivalent oligonucleotides comprising only phosphorothioate internucleoside linkages.

TABLE 115 Plasma triglyceride and APOC-III protein levels in transgenic mice Time point (days APOC-III ISIS Dosage post- Triglycerides protein (% GalNAc₃ No. (mg/kg) dose) (% baseline) baseline) Cluster CM PBS n/a 3 96 101 n/a n/a 7 88 98 14 91 103 21 69 92 28 83 81 35 65 86 42 72 88 304801 30 3 42 46 n/a n/a 7 42 51 14 59 69 21 67 81 28 79 76 35 72 95 42 82 92 663084 10 3 35 28 GalNAc₃-3a A_(d) 7 23 24 14 23 26 21 23 29 28 30 22 35 32 36 42 37 47 679241 10 3 38 30 GalNAc₃- A_(d) 7 31 28 19a 14 30 22 21 36 34 28 48 34 35 50 45 42 72 64

Example 104: Synthesis of Oligonucleotides Comprising a 5′-GalNAc₂ Conjugate

Compound 120 is commercially available, and the synthesis of compound 126 is described in Example 49. Compound 120 (1 g, 2.89 mmol), HBTU (0.39 g, 2.89 mmol), and HOBt (1.64 g, 4.33 mmol) were dissolved in DMF (10 mL. and N,N-diisopropylethylamine (1.75 mL, 10.1 mmol) were added. After about 5 min, aminohexanoic acid benzyl ester (1.36 g, 3.46 mmol) was added to the reaction. After 3 h, the reaction mixture was poured into 100 mL of 1 μM NaHSO4 and extracted with 2×50 mL ethyl acetate. Organic layers were combined and washed with 3×40 mL sat NaHCO₃ and 2×brine, dried with Na₂SO₄, filtered and concentrated. The product was purified by silica gel column chromatography (DCM:EA:Hex, 1:1:1) to yield compound 231. LCMS and NMR were consistent with the structure. Compounds 231 (1.34 g, 2.438 mmol) was dissolved in dichloromethane (10 mL) and trifluoracetic acid (10 mL) was added. After stirring at room temperature for 2 h, the reaction mixture was concentrated under reduced pressure and co-evaporated with toluene (3×10 mL). The residue was dried under reduced pressure to yield compound 232 as the trifuloracetate salt. The synthesis of compound 166 is described in Example 54. Compound 166 (3.39 g, 5.40 mmol) was dissolved in DMF (3 mL). A solution of compound 232 (1.3 g, 2.25 mmol) was dissolved in DMF (3 mL) and N,N-diisopropylethylamine (1.55 mL) was added. The reaction was stirred at room temperature for 30 minutes, then poured into water (80 mL) and the aqueous layer was extracted with EtOAc (2×100 mL). The organic phase was separated and washed with sat. aqueous NaHCO₃ (3×80 mL), 1 μM NaHSO₄ (3×80 mL) and brine (2×80 mL), then dried (Na₂SO₄), filtered, and concentrated. The residue was purified by silica gel column chromatography to yield compound 233. LCMS and NMR were consistent with the structure. Compound 233 (0.59 g, 0.48 mmol) was dissolved in methanol (2.2 mL) and ethyl acetate (2.2 mL). Palladium on carbon (10 wt % Pd/C, wet, 0.07 g) was added, and the reaction mixture was stirred under hydrogen atmosphere for 3 h. The reaction mixture was filtered through a pad of Celite and concentrated to yield the carboxylic acid. The carboxylic acid (1.32 g, 1.15 mmol, cluster free acid) was dissolved in DMF (3.2 mL). To this N,N-diisopropylehtylamine (0.3 mL, 1.73 mmol) and PFPTFA (0.30 mL, 1.73 mmol) were added. After 30 min stirring at room temperature the reaction mixture was poured into water (40 mL) and extracted with EtOAc (2×50 mL). A standard work-up was completed as described above to yield compound 234. LCMS and NMR were consistent with the structure. Oligonucleotide 235 was prepared using the general procedure described in Example 46. The GalNAc₂ cluster portion (GalNAc₂-24_(a)) of the conjugate group GalNAc₂-24 can be combined with any cleavable moiety present on the oligonucleotide to provide a variety of conjugate groups. The structure of GalNAc₂-24 (GalNAc₂-24_(a)-CM) is shown below:

Example 105: Synthesis of Oligonucleotides Comprising a GalNAc₁-25 Conjugate

The synthesis of compound 166 is described in Example 54. Oligonucleotide 236 was prepared using the general procedure described in Example 46.

Alternatively, oligonucleotide 236 was synthesized using the scheme shown below, and compound 238 was used to form the oligonucleotide 236 using procedures described in Example 10.

The GalNAc₁ cluster portion (GalNAc₁-25_(a)) of the conjugate group GalNAc₁-25 can be combined with any cleavable moiety present on the oligonucleotide to provide a variety of conjugate groups. The structure of GalNAc₁-25 (GalNAc₁-25_(a)-CM) is shown below:

Example 106: Antisense Inhibition In Vivo by Oligonucleotides Targeting SRB-1 Comprising a 5′-GalNAc₂ or a 5′-GalNAc₃ Conjugate

Oligonucleotides listed in Tables 116 and 117 were tested in dose-dependent studies for antisense inhibition of SRB-1 in mice.

Treatment

Six to week old, male C57BL/6 mice (Jackson Laboratory, Bar Harbor, Me.) were injected subcutaneously once with 2, 7, or 20 mg/kg of ISIS No. 440762; or with 0.2, 0.6, 2, 6, or 20 mg/kg of ISIS No. 686221, 686222, or 708561; or with saline. Each treatment group consisted of 4 animals. The mice were sacrificed 72 hours following the final administration. Liver SRB-1 mRNA levels were measured using real-time PCR. SRB-1 mRNA levels were normalized to cyclophilin mRNA levels according to standard protocols. The antisense oligonucleotides lowered SRB-1 mRNA levels in a dose-dependent manner, and the ED₅₀ results are presented in Tables 116 and 117. Although previous studies showed that trivalent GalNAc-conjugated oligonucleotides were significantly more potent than divalent GalNAc-conjugated oligonucleotides, which were in turn significantly more potent than monovalent GalNAc conjugated oligonucleotides (see, e.g., Khorev et al., Bioorg. & Med. Chem., Vol. 16, 5216-5231 (2008)), treatment with antisense oligonucleotides comprising monovalent, divalent, and trivalent GalNAc clusters lowered SRB-1 mRNA levels with similar potencies as shown in Tables 116 and 117.

TABLE 116 Modified oligonucleotides targeting SRB-1 ISIS ED₅₀ SEQ No. Sequences (5′ to 3′) GalNAc Cluster (mg/kg) ID No 440762 T_(ks) ^(m)C_(ks)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(ks) ^(m)C_(k) n/a 4.7 4880 686221 GalNAc ₂ -24 _(a) - _(o′) A _(do)T_(ks) ^(m)C_(ks)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) GalNAc₂-24_(a) 0.39 4884 ^(m)C_(ds)T_(ds)T_(ks) ^(m)C_(k) 686222 GalNAc ₃ -13 _(a) - _(o′) A _(do)T_(ks) ^(m)C_(ks)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) GalNAc₃-13_(a) 0.41 4884 ^(m)C_(ds)T_(ds)T_(ks) ^(m)C_(k) See Example 93 for table legend. The structure of GalNAc₃-13a was shown in Example 62, and the structure of GalNAc₂-24a was shown in Example 104.

TABLE 117 Modified oligonucleotides targeting SRB-1 ISIS ED₅₀ SEQ No. Sequences (5′ to 3′) GalNAc Cluster (mg/kg) ID No 440762 T_(ks) ^(m)C_(ks)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(ks) ^(m)C_(k) n/a 5 4880 708561 GalNAc ₁ -25 _(a) - _(o′)T_(ks) ^(m)C_(ks)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) GalNAc₁-25_(a) 0.4 4880 ^(m)C_(ds)T_(ds)T_(ks) ^(m)C_(k) See Example 93 for table legend. The structure of GalNAc₁-25a was shown in Example 105.

The concentrations of the oligonucleotides in Tables 116 and 117 in liver were also assessed, using procedures described in Example 75. The results shown in Tables 117a and 117b below are the average total antisense oligonucleotide tissues levels for each treatment group, as measured by UV in units of μg oligonucleotide per gram of liver tissue. The results show that the oligonucleotides comprising a GalNAc conjugate group accumulated in the liver at significantly higher levels than the same dose of the oligonucleotide lacking a GalNAc conjugate group. Furthermore, the antisense oligonucleotides comprising one, two, or three GalNAc ligands in their respective conjugate groups all accumulated in the liver at similar levels. This result is surprising in view of the Khorev et al. literature reference cited above and is consistent with the activity data shown in Tables 116 and 117 above.

TABLE 117a Liver concentrations of oligonucleotides comprising a GalNAc₂ or GalNAc₃ conjugate group [Antisense Dosage oligonucleotide] GalNAc ISIS No. (mg/kg) (μg/g) cluster CM 440762 2 2.1 n/a n/a 7 13.1 20 31.1 686221 0.2 0.9 GalNAc₂-24_(a) A_(d) 0.6 2.7 2 12.0 6 26.5 686222 0.2 0.5 GalNAc₃-13_(a) A_(d) 0.6 1.6 2 11.6 6 19.8

TABLE 117b Liver concentrations of oligonucleotides comprising a GalNAc₁ conjugate group [Antisense Dosage oligonucleotide] GalNAc ISIS No. (mg/kg) (μg/g) cluster CM 440762 2 2.3 n/a n/a 7 8.9 20 23.7 708561 0.2 0.4 GalNAc₁-25_(a) PO 0.6 1.1 2 5.9 6 23.7 20 53.9

Example 107: Synthesis of Oligonucleotides Comprising a GalNAc₁-26 or GalNAc₁-27 Conjugate

Oligonucleotide 239 is synthesized via coupling of compound 47 (see Example 15) to acid 64 (see Example 32) using HBTU and DIEA in DMF. The resulting amide containing compound is phosphitylated, then added to the 5′-end of an oligonucleotide using procedures described in Example 10. The GalNAc₁ cluster portion (GalNAc₁-26_(a)) of the conjugate group GalNAc₁-26 can be combined with any cleavable moiety present on the oligonucleotide to provide a variety of conjugate groups. The structure of GalNAc₁-26 (GalNAc₁-26_(a)-CM) is shown below:

In order to add the GalNAc₁ conjugate group to the 3′-end of an oligonucleotide, the amide formed from the reaction of compounds 47 and 64 is added to a solid support using procedures described in Example 7. The oligonucleotide synthesis is then completed using procedures described in Example 9 in order to form oligonucleotide 240.

The GalNAc₁ cluster portion (GalNAc₁-27_(a)) of the conjugate group GalNAc₁-27 can be combined with any cleavable moiety present on the oligonucleotide to provide a variety of conjugate groups. The structure of GalNAc₁-27 (GalNAc₁-27_(a)-CM) is shown below:

Example 108: Antisense Inhibition In Vivo by Oligonucleotides Comprising a GalNAc Conjugate Group Targeting Apo(a) In Vivo

The oligonucleotides listed in Table 118 below were tested in a single dose study in mice.

TABLE 118 Modified ASOs targeting APO(a) ISIS GalNAc₃ SEQ No. Sequences (5′ to 3′) Cluster CM ID No. 494372 T_(es)G_(es) ^(m)C_(es)T_(es) ^(m)C_(es) ^(m)C_(ds)G_(ds)T_(ds)T_(ds)G_(ds)G_(ds)T_(ds)G_(ds) ^(m)C_(ds) n/a n/a 4903 T_(ds)T_(es)G_(es)T_(es)T_(es) ^(m)C_(e) 681251 GalNAc ₃ -7 _(a) - _(o′)T_(es)G_(es) ^(m)C_(es)T_(es) ^(m)C_(es) ^(m)C_(ds)G_(ds)T_(ds)T_(ds)G_(ds)G_(ds) GalNAc₃-7a PO 4903 T_(ds)G_(ds) ^(m)C_(ds)T_(ds)T_(es)G_(es)T_(es)T_(es) ^(m)C_(e) 681255 GalNAc ₃ -3 _(a) - _(o′)T_(es)G_(eo) ^(m)C_(eo)T_(eo) ^(m)C_(eo) ^(m)C_(ds)G_(ds)T_(ds)T_(ds)G_(ds)G_(ds) GalNAc₃-3a PO 4903 T_(ds)G_(ds) ^(m)C_(ds)T_(ds)T_(eo)G_(eo)T_(es)T_(es) ^(m)C_(e) 681256 GalNAc ₃ -10 _(a) - _(o′)T_(es)G_(eo) ^(m)C_(eo)T_(eo) ^(m)C_(eo) ^(m)C_(ds)G_(ds)T_(ds)T_(ds)G_(ds)G_(ds) GalNAc₃-10a PO 4903 T_(ds)G_(ds) ^(m)C_(ds)T_(ds)T_(eo)G_(eo)T_(es)T_(es) ^(m)C_(e) 681257 GalNAc ₃ -7 _(a) - _(o′)T_(es)G_(eo) ^(m)C_(eo)T_(eo) ^(m)C_(eo) ^(m)C_(ds)G_(ds)T_(ds)T_(ds)G_(ds)G_(ds) GalNAc₃-7a PO 4903 T_(ds)G_(ds) ^(m)C_(ds)T_(ds)T_(eo)G_(eo)T_(es)T_(es) ^(m)C_(e) 681258 GalNAc ₃ -13 _(a) - _(o′)T_(es)G_(eo) ^(m)C_(eo)T_(eo) ^(m)C_(eo) ^(m)C_(ds)G_(ds)T_(ds)T_(ds)G_(ds)G_(ds) GalNAc₃-13a PO 4903 T_(ds)G_(ds) ^(m)C_(ds)T_(ds)T_(eo)G_(eo)T_(es)T_(es) ^(m)C_(e) 681260 T_(es)G_(eo) ^(m)C_(eo)T_(eo) ^(m)C_(eo) ^(m)C_(ds)G_(ds)T_(ds)T_(ds)G_(ds)G_(ds)T_(ds)G_(ds) ^(m)C_(ds)T_(ds)T_(eo)G_(eo) GalNAc₃-19a A_(d) 4911 T_(es)T_(es) ^(m)C_(eo) A _(do′) -GalNAc ₃ -19

The structure of GalNAc₃-7_(a) was shown in Example 48.

Treatment

Male transgenic mice that express human Apo(a) were each injected subcutaneously once with an oligonucleotide and dosage listed in Table 119 or with PBS. Each treatment group consisted of 4 animals. Blood was drawn the day before dosing to determine baseline levels of Apo(a) protein in plasma and at 1 week following the first dose. Additional blood draws will occur weekly for approximately 8 weeks. Plasma Apo(a) protein levels were measured using an ELISA. The results in Table 119 are presented as the average percent of plasma Apo(a) protein levels for each treatment group, normalized to baseline levels (% BL), The results show that the antisense oligonucleotides reduced Apo(a) protein expression. Furthermore, the oligonucleotides comprising a GalNAc conjugate group exhibited even more potent reduction in Apo(a) expression than the oligonucleotide that does not comprise a conjugate group.

TABLE 119 Apo(a) plasma protein levels Apo(a) at Dosage 1 week ISIS No. (mg/kg) (% BL) PBS n/a 143 494372 50 58 681251 10 15 681255 10 14 681256 10 17 681257 10 24 681258 10 22 681260 10 26

Example 109: Synthesis of Oligonucleotides Comprising a GalNAc₁-28 or GalNAc₁-29 Conjugate

Oligonucleotide 241 is synthesized using procedures similar to those described in Example 71 to form the phosphoramidite intermediate, followed by procedures described in Example 10 to synthesize the oligonucleotide. The GalNAc₁ cluster portion (GalNAc₁-28_(a)) of the conjugate group GalNAc₁-28 can be combined with any cleavable moiety present on the oligonucleotide to provide a variety of conjugate groups. The structure of GalNAc₁-28 (GalNAc₁-28_(a)-CM) is shown below:

In order to add the GalNAc₁ conjugate group to the 3′-end of an oligonucleotide, procedures similar to those described in Example 71 are used to form the hydroxyl intermediate, which is then added to the solid support using procedures described in Example 7. The oligonucleotide synthesis is then completed using procedures described in Example 9 in order to form oligonucleotide 242.

The GalNAc₁ cluster portion (GalNAc₁-29_(a)) of the conjugate group GalNAc₁-29 can be combined with any cleavable moiety present on the oligonucleotide to provide a variety of conjugate groups. The structure of GalNAc₁-29 (GalNAc₁-29_(a)-CM) is shown below:

Example 110: Synthesis of Oligonucleotides Comprising a GalNAc₁-30 Conjugate

Oligonucleotide 246 comprising a GalNAc₁-30 conjugate group, wherein Y is selected from O, S, a substituted or unsubstituted C₁-C₁₀ alkyl, amino, substituted amino, azido, alkenyl or alkynyl, is synthesized as shown above. The GalNAc₁ cluster portion (GalNAc₁-30_(a)) of the conjugate group GalNAc₁-30 can be combined with any cleavable moiety to provide a variety of conjugate groups. In certain embodiments, Y is part of the cleavable moiety. In certain embodiments, Y is part of a stable moiety, and the cleavable moiety is present on the oligonucleotide. The structure of GalNAc₁-30_(a) is shown below:

Example 111: Synthesis of Oligonucleotides Comprising a GalNAc₂-31 or GalNAc₂-32 Conjugate

Oligonucleotide 250 comprising a GalNAc₂-31 conjugate group, wherein Y is selected from O, S, a substituted or unsubstituted C₁-C₁₀ alkyl, amino, substituted amino, azido, alkenyl or alkynyl, is synthesized as shown above. The GalNAc₂ cluster portion (GalNAc₂-31a) of the conjugate group GalNAc₂-31 can be combined with any cleavable moiety to provide a variety of conjugate groups. In certain embodiments, the Y-containing group directly adjacent to the 5′-end of the oligonucleotide is part of the cleavable moiety. In certain embodiments, the Y-containing group directly adjacent to the 5′-end of the oligonucleotide is part of a stable moiety, and the cleavable moiety is present on the oligonucleotide. The structure of GalNAc₂-31a is shown below:

The synthesis of an oligonucleotide comprising a GalNAc₂-32 conjugate is shown below.

Oligonucleotide 252 comprising a GalNAc₂-32 conjugate group, wherein Y is selected from O, S, a substituted or unsubstituted C₁-C₁₀ alkyl, amino, substituted amino, azido, alkenyl or alkynyl, is synthesized as shown above. The GalNAc₂ cluster portion (GalNAc₂-32_(a)) of the conjugate group GalNAc₂-32 can be combined with any cleavable moiety to provide a variety of conjugate groups. In certain embodiments, the Y-containing group directly adjacent to the 5′-end of the oligonucleotide is part of the cleavable moiety. In certain embodiments, the Y-containing group directly adjacent to the 5′-end of the oligonucleotide is part of a stable moiety, and the cleavable moiety is present on the oligonucleotide. The structure of GalNAc₂-32_(a) is shown below:

Example 112: Modified Oligonucleotides Comprising a GalNAc₁ Conjugate

The oligonucleotides in Table 120 targeting SRB-1 were synthesized with a GalNAc₁ conjugate group in order to further test the potency of oligonucleotides comprising conjugate groups that contain one GalNAc ligand.

TABLE 120 GalNAc SEQ ISIS No. Sequence (5′ to 3′) cluster CM ID NO. 711461 GalNAc ₁ -25 _(a-o′) A _(do) G_(es) ^(m)C_(es) T_(es) T_(es) ^(m)C_(es) A_(ds) G_(ds) T_(ds) ^(m)C_(ds) A_(ds) GalNAc₁-25_(a) A_(d) 4888 T_(ds) G_(ds) A_(ds) ^(m)C_(ds) T_(ds) T_(es) ^(m)C_(es) ^(m)C_(es) T_(es) T_(e) 711462 GalNAc ₁ -25 _(a-o′)G_(es) ^(m)C_(es) T_(es) T_(es) ^(m)C_(es) A_(ds) G_(ds) T_(ds) ^(m)C_(ds) A_(ds) T_(ds) GalNAc₁-25_(a) PO 4886 G_(ds) A_(ds) ^(m)C_(ds) T_(ds) T_(es) ^(m)C_(es) ^(m)C_(es) T_(es) T_(e) 711463 GalNAc ₁ -25 _(a-o′)G_(es) ^(m)C_(eo) T_(eo) T_(eo) ^(m)C_(eo) A_(ds) G_(ds) T_(ds) ^(m)C_(ds) A_(ds) T_(ds) GalNAc₁-25_(a) PO 4886 G_(ds) A_(ds) ^(m)C_(ds) T_(ds) T_(eo) ^(m)C_(eo) ^(m)C_(es) T_(es) T_(e) 711465 GalNAc ₁ -26 _(a-o′) A _(do) G_(es) ^(m)C_(es) T_(es) T_(es) ^(m)C_(es) A_(ds) G_(ds) T_(ds) ^(m)C_(ds) A_(ds) T_(ds) GalNAc₁-26_(a) A_(d) 4888 G_(ds) A_(ds) ^(m)C_(ds) T_(ds) T_(es) ^(m)C_(es) ^(m)C_(es) T_(es) T_(e) 711466 GalNAc ₁ -26 _(a-o′)G_(es) ^(m)C_(es) T_(es) T_(es) ^(m)C_(es) A_(ds) G_(ds) T_(ds) ^(m)C_(ds) A_(ds) T_(ds) GalNAc₁-26_(a) PO 4886 G_(ds) A_(ds) ^(m)C_(ds) T_(ds) T_(es) ^(m)C_(es) ^(m)C_(es) T_(es) T_(e) 711467 GalNAc ₁ -26 _(a-o′)G_(es) ^(m)C_(eo) T_(eo) T_(eo) ^(m)C_(eo) A_(ds) G_(ds) T_(ds) ^(m)C_(ds) A_(ds) T_(ds) GalNAc₁-26_(a) PO 4886 G_(ds) A_(ds) ^(m)C_(ds) T_(ds) T_(eo) ^(m)C_(eo) ^(m)C_(es) T_(es) T_(e) 711468 GalNAc ₁ -28 _(a-o′) A _(do) G_(es) ^(m)C_(es) T_(es) T_(es) ^(m)C_(es) A_(ds) G_(ds) T_(ds) ^(m)C_(ds) A_(ds) GalNAc₁-28_(a) A_(d) 4888 T_(ds) G_(ds) A_(ds) ^(m)C_(ds) T_(ds) T_(es) ^(m)C_(es) ^(m)C_(es) T_(es) T_(e) 711469 GalNAc ₁ -28 _(a-o′)G_(es) ^(m)C_(es) T_(es) T_(es) ^(m)C_(es) A_(ds) G_(ds) T_(ds) ^(m)C_(ds) A_(ds) T_(ds) GalNAc₁-28_(a) PO 4886 G_(ds) A_(ds) ^(m)C_(ds) T_(ds) T_(es) ^(m)C_(es) ^(m)C_(es) T_(es) T_(e) 711470 GalNAc ₁ -28 _(a-o′)G_(es) ^(m)C_(eo) T_(eo) T_(eo) ^(m)C_(eo) A_(ds) G_(ds) T_(ds) ^(m)C_(ds) A_(ds) T_(ds) GalNAc₁-28_(a) PO 4886 G_(ds) A_(ds) ^(m)C_(ds) T_(ds) T_(eo) ^(m)C_(eo) ^(m)C_(es) T_(es) T_(e) 713844 G_(es) ^(m)C_(es) T_(es) T_(es) ^(m)C_(es) A_(ds) G_(ds) T_(ds) ^(m)C_(ds) A_(ds) T_(ds) G_(ds) A_(ds) ^(m)C_(ds) T_(ds) GalNAc₁-27_(a) PO 4886 T_(es) ^(m)C_(es) ^(m)C_(es) T_(es) T_(eo′-) GalNAc ₁ -27 _(a) 713845 G_(es) ^(m)C_(eo) T_(eo) T_(eo) ^(m)C_(eo) A_(ds) G_(ds) T_(ds) ^(m)C_(ds) A_(ds) T_(ds) G_(ds) A_(ds) ^(m)C_(ds) T_(ds) GalNAc₁-27_(a) PO 4886 T_(eo) ^(m)C_(eo) ^(m)C_(es) T_(es) T_(eo′-) GalNAc ₁ -27 _(a) 713846 G_(es) ^(m)C_(eo) T_(eo) T_(eo) ^(m)C_(eo) A_(ds) G_(ds) T_(ds) ^(m)C_(ds) A_(ds) T_(ds) G_(ds) A_(ds) ^(m)C_(ds) T_(ds) GalNAc₁-27_(a) A_(d) 4887 T_(eo) ^(m)C_(eo) ^(m)C_(es) T_(es) T_(eo) A _(do′-) GalNAc ₁ -27 _(a) 713847 G_(es) ^(m)C_(es) T_(es) T_(es) ^(m)C_(es) A_(ds) G_(ds) T_(ds) ^(m)C_(ds) A_(ds) T_(ds) G_(ds) A_(ds) ^(m)C_(ds) T_(ds) GalNAc₁-29_(a) PO 4886 T_(es) ^(m)C_(es) ^(m)C_(es) T_(es) T_(eo′-) GalNAc ₁ -29 _(a) 713848 G_(es) ^(m)C_(eo) T_(eo) T_(eo) ^(m)C_(eo) A_(ds) G_(ds) T_(ds) ^(m)C_(ds) A_(ds) T_(ds) G_(ds) A_(ds) ^(m)C_(ds) T_(ds) GalNAc₁-29_(a) PO 4886 T_(eo) ^(m)C_(eo) ^(m)C_(es) T_(es) T_(eo′-) GalNAc ₁ -29 _(a) 713849 G_(es) ^(m)C_(es) T_(es) T_(es) ^(m)C_(es) A_(ds) G_(ds) T_(ds) ^(m)C_(ds) A_(ds) T_(ds) G_(ds) A_(ds) ^(m)C_(ds) T_(ds) GalNAc₁-29_(a) A_(d) 4887 T_(es) ^(m)C_(es) ^(m)C_(es) T_(es) T_(eo) A _(do′-) GalNAc ₁ -29 _(a) 713850 G_(es) ^(m)C_(eo) T_(eo) T_(eo) ^(m)C_(eo) A_(ds) G_(ds) T_(ds) ^(m)C_(ds) A_(ds) T_(ds) G_(ds) A_(ds) ^(m)C_(ds) T_(ds) GalNAc₁-29_(a) A_(d) 4887 T_(eo) ^(m)C_(eo) ^(m)C_(es) T_(es) T_(eo) A _(do′-) GalNAc ₁ -29 _(a)

Example 113: Antisense Oligonucleotides Targeting Angiopoietin-Like 3 and Comprising a GalNAc Conjugate Group

The oligonucleotides in Table 121 were designed to target human angiopoietin-like 3 (ANGPTL3).

TABLE 121 ISIS SEQ No. Sequences (5′ to 3′) ID No. 563580 G_(es)G_(es)A_(es) ^(m)C_(es)A_(es)T_(ds)T_(ds)G_(ds) ^(m)C_(ds) ^(m)C_(ds)A_(ds)G_(ds)T_(ds)A_(ds)A_(ds)T_(es) ^(m)C_(es)G_(es) ^(m)C_(es)A_(e) 77 (parent) 658501 G_(es)G_(es)A_(es) ^(m)C_(es)A_(es)T_(ds)T_(ds)G_(ds) ^(m)C_(ds) ^(m)C_(ds)A_(ds)G_(ds)T_(ds)A_(ds)A_(ds)T_(es) ^(m)C_(es)G_(es) ^(m)C_(es)A_(eo) A _(do′) -GalNAc ₃ -1 _(a) 4912 666944 GalNAc ₃ -3 _(a-o′) A _(do)G_(es)G_(es)A_(es) ^(m)C_(es)A_(es)T_(ds)T_(ds)G_(ds) ^(m)C_(ds) ^(m)C_(ds)A_(ds)G_(ds)T_(ds)A_(ds)A_(ds)T_(es) ^(m)C_(es)G_(es) ^(m)C_(es)A_(e) 4913 666945 G_(es)G_(eo)A_(eo) ^(m)C_(eo)A_(eo)T_(ds)T_(ds)G_(ds) ^(m)C_(ds) ^(m)C_(ds)A_(ds)G_(ds)T_(ds)A_(ds)A_(ds)T_(eo) ^(m)C_(eo)G_(es) ^(m)C_(es)A_(eo) A _(do′) -GalNAc ₃ -1 _(a) 4912 666946 GalNAc ₃ -3 _(a-o′) A _(do)G_(es)G_(eo)A_(eo) ^(m)C_(eo)A_(eo)T_(ds)T_(ds)G_(ds) ^(m)C_(ds) ^(m)C_(ds)A_(ds)G_(ds)T_(ds)A_(ds)A_(ds)T_(eo) ^(m)C_(eo)G_(es) ^(m)C_(es)A_(e) 4913 703801 GalNAc ₃ -7 _(a-o′)G_(es)G_(es)A_(es) ^(m)C_(es)A_(es)T_(ds)T_(ds)G_(ds) ^(m)C_(ds) ^(m)C_(ds)A_(ds)G_(ds)T_(ds)A_(ds)A_(ds)T_(es) ^(m)C_(es)G_(es) ^(m)C_(es)A_(e) 77 703802 GalNAc ₃ -7 _(a-o′)G_(es)G_(eo)A_(eo) ^(m)C_(eo)A_(eo)T_(ds)T_(ds)G_(ds) ^(m)C_(ds) ^(m)C_(ds)A_(ds)G_(ds)T_(ds)A_(ds)A_(ds)T_(eo) ^(m)C_(eo)G_(es) ^(m)C_(es)A_(e) 77

Example 114: Antisense Inhibition In Vivo by Oligonucleotides Comprising a GalNAc Conjugate Group Targeting Human ANGPTL3

Six week old male, transgenic C57Bl/6 mice that express human ANGPTL3 were each injected intraperitoneally once per week at a dosage shown below, for a total of two doses, with an oligonucleotide listed in Table 122 (and described in Table 121) or with PBS. Each treatment group consisted of 4 animals. The mice were sacrificed two days following the final dose. ANGPTL3 liver mRNA levels were measured using real-time PCR and RIBOGREEN RNA quantification reagent (Molecular Probes, Inc. Eugene, Oreg.) according to standard protocols. The results below are presented as the average percent of ANGPTL3 mRNA levels in liver for each treatment group, normalized to the PBS control.

As illustrated in Table 122, treatment with antisense oligonucleotides lowered ANGPTL3 liver mRNA levels in a dose-dependent manner, and the oligonucleotide comprising a GalNAc conjugate was significantly more potent than the parent oligonucleotide lacking a GalNAc conjugate.

TABLE 122 ANGPTL3 liver mRNA levels Dosage mRNA GalNAc₃ ISIS No. (mg/kg) (% PBS) Cluster CM 563580 5 58 n/a n/a 10 56 15 36 25 23 50 20 658501 0.3 78 GalNAc₃-1a A_(d) 1 60 3 27 10 19

Liver alanine aminotransferase (ALT) levels were also measured at time of sacrifice using standard protocols. The results are showed that none of the treatment groups had elevated ALT levels, indicating that the oligonucleotides were well tolerated.

Example 115: Antisense Inhibition In Vivo by Oligonucleotides Comprising a GalNAc Conjugate Group Targeting Mouse ANGPTL3

The oligonucleotides listed in Table 123 below were tested in a dose-dependent study in mice.

TABLE 123 Modified ASOs targeting mouse ANGPTL3 ISIS GalNAc₃ SEQ No. Sequences (5′ to 3′) Cluster CM ID No. 233693 G_(es)A_(es) ^(m)C_(es)A_(es)T_(es)G_(ds)T_(ds)T_(ds) ^(m)C_(ds)T_(ds)T_(ds) ^(m)C_(ds)A_(ds) ^(m)C_(ds) n/a n/a 4914 ^(m)C_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es) ^(m)C_(e) 703803 GalNAc ₃ -7 _(a)-_(o′)G_(es)A_(es) ^(m)C_(es)A_(es)T_(es)G_(ds)T_(ds)T_(ds) ^(m)C_(ds)T_(ds)T_(ds) GalNAc₃-7a PO 4914 ^(m)C_(ds)A_(ds) ^(m)C_(ds) ^(m)C_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es) ^(m)C_(e) 703804 GalNAc ₃ -7 _(a)-_(o′)G_(es)A_(eo) ^(m)C_(eo)A_(eo)T_(eo)G_(ds)T_(ds)T_(ds) ^(m) ^(C) _(ds)T_(ds)T_(ds) GalNAc₃-7a PO 4914 ^(m)C_(ds)A_(ds) ^(m)C_(ds) ^(m)C_(ds)T_(eo) ^(m)C_(eo) ^(m)C_(es)T_(es) ^(m)C_(e)

The structure of GalNAc₃-7a was shown in Example 48. Low density lipoprotein receptor knock-out (LDLR^(−/−)) mice were fed a western diet for 1 week before being injected intraperitoneally once per week at a dosage shown below with an oligonucleotide listed in Table 123 or with PBS. Each treatment group consisted of 5 animals. Blood was drawn before the first dose was administered in order to determine baseline levels oftriglycerides in plasma and at 2 weeks following the first dose. The results in Table 124 are presented as the average percent of plasma triglyceride levels for each treatment group, normalized to baseline levels (% BL), The results show that the antisense oligonucleotides reduced triglycerides in a dose dependent manner. Furthermore, the oligonucleotides comprising a GalNAc conjugate group exhibited even more potent reduction in triglycerides than the oligonucleotide that does not comprise a conjugate group.

TABLE 124 Plasma triglyceride (TG) levels Dosage TG ED₅₀ GalNAc₃ ISIS No. (mg/kg) (% BL) (mg/kg) Cluster CM PBS n/a 110 n/a n/a n/a 233693 1 92 16 n/a n/a 3 71 10 57 30 42 703803 0.3 96 2 GalNAc₃ ₋ 7a PO 1 69 3 39 10 27 703804 0.3 97 2 GalNAc₃ ₋ 7a PO 1 54 3 38 10 26

Example 116: Antisense Inhibition of Human Angiopoietin-Like 3 in Hep3B Cells by MOE Gapmers

Antisense oligonucleotides were designed targeting an Angiopoietin-like 3 (ANGPTL3) nucleic acid and were tested for their effects on ANGPTL3 mRNA in vitro. The antisense oligonucleotides were tested in a series of experiments that had similar culture conditions. The results for each experiment are presented in separate tables shown below. Cultured Hep3B cells at a density of 20,000 cells per well were transfected using electroporation with 4,500 nM antisense oligonucleotide. After a treatment period of approximately 24 hours, RNA was isolated from the cells and ANGPTL3 mRNA levels were measured by quantitative real-time PCR. Human primer probe set RTS3492_MGB (forward sequence CCGTGGAAGACCAATATAAACAATT, designated herein as SEQ ID NO: 4; AGTCCTTCTGAGCTGATTTTCTATTTCT; reverse sequence, designated herein as SEQ ID NO: 5; probe sequence AACCAACAGCATAGTCAAATA, designated herein as SEQ ID NO: 6) was used to measure mRNA levels. ANGPTL3 mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of ANGPTL3, relative to untreated control cells.

The newly designed chimeric antisense oligonucleotides in the Tables below were designed as 5-10-5 MOE gapmers. The 5-10-5 MOE gapmers are 20 nucleosides in length, wherein the central gap segment comprises often 2′-deoxynucleosides and is flanked by wing segments on the 5′ direction and the 3′ direction comprising five nucleosides each. Each nucleoside in the 5′ wing segment and each nucleoside in the 3′ wing segment has a 2′-MOE modification. The internucleoside linkages throughout each gapmer are phosphorothioate (P═S) linkages. All cytosine residues throughout each gapmer are 5-methylcytosines. “Start site” indicates the 5′-most nucleoside to which the gapmer is targeted in the human gene sequence. “Stop site” indicates the 3′-most nucleoside to which the gapmer is targeted human gene sequence. Each gapmer listed in the Tables below is targeted to either the human ANGPTL3 mRNA, designated herein as SEQ ID NO: 1 (GENBANK Accession No. NM_014495.2) or the human ANGPTL3 genomic sequence, designated herein as SEQ ID NO: 2 (GENBANK Accession No. NT_032977.9 truncated from nucleotides 33032001 to 33046000). ‘n/a’ indicates that the antisense oligonucleotide does not target that particular gene sequence with 100% complementarity.

TABLE 125 Inhibition of ANGPTL3 mRNA by 5-10-5 MOE gapmers targeting SEQ ID NO: 1 and 2 SEQ SEQ SEQ SEQ ID ID ID ID NO: 1 NO: 1 NO: 2 NO: 2 SEQ ISIS Start Stop % Start Stop ID NO Site Site Sequence inhibition Site Site NO 544059 23 42 GATTTTCAATTTCAAGCAAC 40 3127 3146 238 337459 49 68 AGCTTAATTGTGAACATTTT 47 3153 3172 239 544060 54 73 GAAGGAGCTTAATTGTGAAC 1 3158 3177 240 544061 63 82 CAATAAAAAGAAGGAGCTTA 37 3167 3186 241 544062 66 85 GAACAATAAAAAGAAGGAGC 38 3170 3189 242 544063 85 104 CTGGAGGAAATAACTAGAGG 30 3189 3208 243 337460 88 107 ATTCTGGAGGAAATAACTAG 39 3192 3211 244 544064 112 131 TCAAATGATGAATTGTCTTG 36 3216 3235 245 544065 138 157 TTGATTTTGGCTCTGGAGAT 26 3242 3261 246 544066 145 164 GCAAATCTTGATTTTGGCTC 56 3249 3268 247 233676 148 167 ATAGCAAATCTTGATTTTGG 69 3252 3271 248 544067 156 175 CGTCTAACATAGCAAATCTT 64 3260 3279 249 544068 174 193 TGGCTAAAATTTTTACATCG 28 3278 3297 250 544069 178 197 CCATTGGCTAAAATTTTTAC 0 3282 3301 251 544070 184 203 AGGAGGCCATTGGCTAAAAT 7 3288 3307 252 544071 187 206 TGAAGGAGGCCATTGGCTAA 32 3291 3310 253 544072 195 214 GTCCCAACTGAAGGAGGCCA 9 3299 3318 254 544073 199 218 CCATGTCCCAACTGAAGGAG 6 3303 3322 255 544074 202 221 AGACCATGTCCCAACTGAAG 18 3306 3325 256 544075 206 225 TTTAAGACCATGTCCCAACT 0 3310 3329 257 544076 209 228 GTCTTTAAGACCATGTCCCA 0 3313 3332 258 544077 216 235 GGACAAAGTCTTTAAGACCA 0 3320 3339 259 544078 222 241 TCTTATGGACAAAGTCTTTA 0 3326 3345 260 544079 245 264 TATGTCATTAATTTGGCCCT 0 3349 3368 261 544080 270 289 GATCAAATATGTTGAGTTTT 27 3374 3393 262 233690 274 293 GACTGATCAAATATGTTGAG 49 3378 3397 263 544081 316 335 TCTTCTTTGATTTCACTGGT 62 3420 3439 264 544082 334 353 CTTCTCAGTTCCTTTTCTTC 35 3438 3457 265 544083 337 356 GTTCTTCTCAGTTCCTTTTC 60 3441 3460 266 544084 341 360 TGTAGTTCTTCTCAGTTCCT 51 3445 3464 267 544431 345 364 TATATGTAGTTCTTCTCAGT 9 3449 3468 268 544086 348 367 GTTTATATGTAGTTCTTCTC 39 3452 3471 269 544087 352 371 TGTAGTTTATATGTAGTTCT 30 3456 3475 270 544088 356 375 GACTTGTAGTTTATATGTAG 12 3460 3479 271 544089 364 383 TCATTTTTGACTTGTAGTTT 31 3468 3487 272 544090 369 388 CCTCTTCATTTTTGACTTGT 61 3473 3492 273 544091 375 394 TCTTTACCTCTTCATTTTTG 48 3479 3498 274 544092 380 399 CATATTCTTTACCTCTTCAT 35 3484 3503 275 544093 384 403 GTGACATATTCTTTACCTCT 63 3488 3507 276 544094 392 411 GAGTTCAAGTGACATATTCT 53 3496 3515 277 544095 398 417 TGAGTTGAGTTCAAGTGACA 31 3502 3521 278 544096 403 422 AGTTTTGAGTTGAGTTCAAG 14 3507 3526 279 544097 406 425 TCAAGTTTTGAGTTGAGTTC 38 3510 3529 280 544098 414 433 GGAGGCTTTCAAGTTTTGAG 39 3518 3537 281 544099 423 442 TTTCTTCTAGGAGGCTTTCA 57 3527 3546 282 544100 427 446 ATTTTTTCTTCTAGGAGGCT 39 3531 3550 283 544101 432 451 GTAGAATTTTTTCTTCTAGG 28 3536 3555 284 544102 462 481 GCTCTTCTAAATATTTCACT 60 3566 3585 285 544103 474 493 AGTTAGTTAGTTGCTCTTCT 40 3578 3597 286 544104 492 511 CAGGTTGATTTTGAATTAAG 38 3596 3615 287 544105 495 514 TTTCAGGTTGATTTTGAATT 28 3599 3618 288 544106 499 518 GGAGTTTCAGGTTGATTTTG 38 3603 3622 289 544107 504 523 GTTCTGGAGTTTCAGGTTGA 50 3608 3627 290 544108 526 545 TTAAGTGAAGTTACTTCTGG 20 3630 3649 291 544109 555 574 TGCTATTATCTTGTTTTTCT 23 4293 4312 292 544110 564 583 GGTCTTTGATGCTATTATCT 67 4302 4321 293 544111 567 586 GAAGGTCTTTGATGCTATTA 49 4305 4324 294 544112 572 591 CTGGAGAAGGTCTTTGATGC 52 4310 4329 295 544113 643 662 CTGAGCTGATTTTCTATTTC 12 n/a n/a 296 337477 664 683 GGTTCTTGAATACTAGTCCT 70 6677 6696 234 544114 673 692 ATTTCTGTGGGTTCTTGAAT 32 6686 6705 297 337478 675 694 AAATTTCTGTGGGTTCTTGA 51 6688 6707 235 544115 678 697 GAGAAATTTCTGTGGGTTCT 54 6691 6710 298 544116 682 701 GATAGAGAAATTTCTGTGGG 25 6695 6714 299 544117 689 708 CTTGGAAGATAGAGAAATTT 16 6702 6721 300 337479 692 711 TGGCTTGGAAGATAGAGAAA 34 6705 6724 236 544118 699 718 GTGCTCTTGGCTTGGAAGAT 64 6712 6731 301 544119 703 722 CTTGGTGCTCTTGGCTTGGA 70 6716 6735 302 544120 707 726 AGTTCTTGGTGCTCTTGGCT 82 6720 6739 15 233710 710 729 AGTAGTTCTTGGTGCTCTTG 63 6723 6742 233 544121 713 732 GGGAGTAGTTCTTGGTGCTC 64 6726 6745 303 544122 722 741 CTGAAGAAAGGGAGTAGTTC 24 6735 6754 304 544123 752 771 ATCATGTTTTACATTTCTTA 0 6765 6784 305 544124 755 774 GCCATCATGTTTTACATTTC 35 n/a n/a 306 544125 759 778 GAATGCCATCATGTTTTACA 8 n/a n/a 307 544126 762 781 CAGGAATGCCATCATGTTTT 6 n/a n/a 308 337487 804 823 CACTTGTATGTTCACCTCTG 65 7389 7408 28 233717 889 908 TGAATTAATGTCCATGGACT 33 7876 7895 14

TABLE 126 Inhibition of ANGPTL3 mRNA by 5-10-5 MOE gapmers targeting SEQ ID NO: 1 and 2 SEQ SEQ SEQ SEQ ID ID ID ID NO: 1 NO: 1 NO: 2 NO: 2 SEQ ISIS Start Stop % Start Stop ID NO Site Site Sequence inhibition Site Site NO 544204 n/a n/a GACTTCTTAACTCTATATAT 0 3076 3095 309 544205 n/a n/a CTAGACTTCTTAACTCTATA 0 3079 3098 310 544206 n/a n/a GACCTAGACTTCTTAACTCT 0 3082 3101 311 544207 n/a n/a GGAAGCAGACCTAGACTTCT 21 3089 3108 312 544208 n/a n/a TCTGGAAGCAGACCTAGACT 23 3092 3111 313 544209 n/a n/a TCTTCTGGAAGCAGACCTAG 7 3095 3114 314 544210 n/a n/a CTAATCTTTAGGGATTTAGG 24 11433 11452 315 544211 n/a n/a TGTATCTAATCTTTAGGGAT 2 11438 11457 316 544213 n/a n/a TAACTTGGGCACTATATCCT 44 11553 11572 317 544214 n/a n/a ATTGACAAAGGTAGGTCACC 59 11576 11595 318 544215 n/a n/a ATATGACATGTATATTGGAT 41 11620 11639 319 544216 n/a n/a TTTTGTACTTTTCTGGAACA 34 11704 11723 320 544217 n/a n/a TAGTCTGTGGTCCTGAAAAT 32 11748 11767 321 544218 n/a n/a AGCTTAGTCTGTGGTCCTGA 20 11752 11771 322 544219 n/a n/a GACAGCTTAGTCTGTGGTCC 45 11755 11774 323 544220 n/a n/a GTATTCTGGCCCTAAAAAAA 2 11789 11808 324 544221 n/a n/a ATTTTGGTATTCTGGCCCTA 39 11795 11814 325 544223 n/a n/a TTTGCATTTGAAATTGTCCA 32 11837 11856 326 544224 n/a n/a GGAAGCAACTCATATATTAA 39 11869 11888 327 544225 n/a n/a TATCAGAAAAAGATACCTGA 0 9821 9840 328 544226 n/a n/a ATAATAGCTAATAATGTGGG 15 9875 9894 329 544227 n/a n/a TGCAGATAATAGCTAATAAT 31 9880 9899 330 544228 n/a n/a TGTCATTGCAGATAATAGCT 61 9886 9905 331 544229 n/a n/a TAAAAGTTGTCATTGCAGAT 38 9893 9912 332 544230 n/a n/a CGGATTTTTAAAAGTTGTCA 45 9901 9920 333 544231 n/a n/a GGGATTCGGATTTTTAAAAG 0 9907 9926 334 544232 n/a n/a TTTGGGATTCGGATTTTTAA 24 9910 9929 335 544233 n/a n/a ACGCTTATTTGGGATTCGGA 53 9917 9936 336 544251 n/a n/a TTTAAGAGATTTACAAGTCA 11 2811 2830 337 544252 n/a n/a GACTACCTGTTTTTAAAAGC 6 2851 2870 338 544253 n/a n/a TATGGTGACTACCTGTTTTT 12 2857 2876 339 544254 n/a n/a ACTTTGCTGTATTATAAACT 12 2890 2909 340 544255 n/a n/a ATTGTATTTAACTTTGCTGT 0 2900 2919 341 544256 n/a n/a GAGCAACTAACTTAATAGGT 13 2928 2947 342 544257 n/a n/a GAAATGAGCAACTAACTTAA 25 2933 2952 343 544258 n/a n/a AATCAAAGAAATGAGCAACT 0 2940 2959 344 544259 n/a n/a ACCTTCTTCCACATTGAGTT 8 2977 2996 345 544260 n/a n/a CACGAATGTAACCTTCTTCC 0 2987 3006 346 544261 n/a n/a TTAACTTGCACGAATGTAAC 27 2995 3014 347 544262 n/a n/a TATATATACCAATATTTGCC 0 3063 3082 348 544263 n/a n/a TCTTAACTCTATATATACCA 0 3072 3091 349 544264 n/a n/a CTTTAAGTGAAGTTACTTCT 17 3632 3651 350 544265 n/a n/a TCTACTTACTTTAAGTGAAG 9 3640 3659 351 544266 n/a n/a GAACCCTCTTTATTTTCTAC 1 3655 3674 352 544267 n/a n/a ACATAAACATGAACCCTCTT 6 3665 3684 353 544268 n/a n/a CCACATTGAAAACATAAACA 25 3676 3695 354 544269 n/a n/a GCATGCCTTAGAAATATTTT 7 3707 3726 355 544270 n/a n/a CAATGCAACAAAGTATTTCA 0 3731 3750 356 544271 n/a n/a CTGGAGATTATTTTTCTTGG 34 3768 3787 357 544272 n/a n/a TTCATATATAACATTAGGGA 0 3830 3849 358 544273 n/a n/a TCAGTGTTTTCATATATAAC 18 3838 3857 359 544274 n/a n/a GACATAGTGTTCTAGATTGT 14 3900 3919 360 544275 n/a n/a CAATAGTGTAATGACATAGT 21 3912 3931 361 544276 n/a n/a TTACTTACCTTCAGTAATTT 0 3933 3952 362 544277 n/a n/a ATCTTTTCCATTTACTGTAT 8 4005 4024 363 544278 n/a n/a AGAAAAAGCCCAGCATATTT 11 4037 4056 364 544279 n/a n/a GTATGCTTCTTTCAAATAGC 36 4130 4149 365 544280 n/a n/a CCTTCCCCTTGTATGCTTCT 41 4140 4159 366 544281 n/a n/a CCTGTAACACTATCATAATC 1 4207 4226 367 544282 n/a n/a TGACTTACCTGATTTTCTAT 6 4384 4403 368 544283 n/a n/a GATGGGACATACCATTAAAA 0 4407 4426 369 544284 n/a n/a GTGAAAGATGGGACATACCA 20 4413 4432 370 544285 n/a n/a CCTGTGTGAAAGATGGGACA 6 4418 4437 371 544286 n/a n/a CATTGGCTGCTATGAATTAA 41 4681 4700 372 544287 n/a n/a GATGACATTGGCTGCTATGA 40 4686 4705 373 544288 n/a n/a GAGAAACATGATCTAATTTG 12 4717 4736 374 544289 n/a n/a ATGGAAAGCTATTGTGTGGT 0 4747 4766 375 544290 n/a n/a GTCTAAAGAGCCAATATGAG 22 4771 4790 376 544291 n/a n/a AATCTTGGTCTAAAGAGCCA 46 4778 4797 377 544433 n/a n/a GAGATTTACAAGTCAAAAAT 4 2806 2825 378 544434 n/a n/a ATTTAACTTTGCTGTATTAT 0 2895 2914 379 544435 n/a n/a ATCAATGCTAAATGAAATCA 0 2955 2974 380 544436 n/a n/a TATTTTCTGGAGATTATTTT 0 3774 3793 381 544437 n/a n/a AAAATGAATATTGGCAATTC 0 4159 4178 382 233717 889 908 TGAATTAATGTCCATGGACT 36 7876 7895 14 544202 2081 2100 AAAGTCAATGTGACTTAGTA 42 11053 11072 383 544203 2104 2123 AAGGTATAGTGATACCTCAT 56 11076 11095 384

TABLE 127 Inhibition of ANGPTL3 mRNA by 5-10-5 MOE gapmers targeting SEQ ID NO: 1 and 2 SEQ ID SEQ ID SEQ ID SEQ ID NO: 1 NO: NO: 2 NO: 2 SEQ ISIS Start 1 Stop % Start Stop ID NO Site Site Sequence inhibition Site Site NO 544127 765 784 CAGCAGGAATGCCATCATGT 4 N/A N/A 385 544128 819 838 TGATGGCATACATGCCACTT 0 7404 7423 386 544129 828 847 TGCTGGGTCTGATGGCATAC 44 7413 7432 387 544130 832 851 GAGTTGCTGGGTCTGATGGC 16 7417 7436 388 544131 841 860 AAAACTTGAGAGTTGCTGGG 0 7426 7445 389 544132 848 867 GACATGAAAAACTTGAGAGT 0 7433 7452 390 544133 859 878 ACATCACAGTAGACATGAAA 25 7444 7463 391 233717 889 908 TGAATTAATGTCCATGGACT 36 7876 7895 14 544134 915 934 AGTTTTGTGATCCATCTATT 46 7902 7921 392 544135 918 937 TGAAGTTTTGTGATCCATCT 42 7905 7924 393 544136 926 945 CGTTTCATTGAAGTTTTGTG 45 7913 7932 394 544137 946 965 CCATATTTGTAGTTCTCCCA 44 7933 7952 395 544138 949 968 AAACCATATTTGTAGTTCTC 25 7936 7955 396 544139 970 989 AATTCTCCATCAAGCCTCCC 35 N/A N/A 397 233722 991 1010 ATCTTCTCTAGGCCCAACCA 65 9566 9585 398 544432 997 1016 GAGTATATCTTCTCTAGGCC 0 9572 9591 399 544140 1002 1021 CTATGGAGTATATCTTCTCT 6 9577 9596 400 544141 1008 1027 GCTTCACTATGGAGTATATC 63 9583 9602 401 544142 1013 1032 AGATTGCTTCACTATGGAGT 52 9588 9607 402 544143 1046 1065 CCAGTCTTCCAACTCAATTC 35 9621 9640 403 544144 1052 1071 GTCTTTCCAGTCTTCCAACT 64 9627 9646 404 544145 1055 1074 GTTGTCTTTCCAGTCTTCCA 80 9630 9649 16 544146 1059 1078 GTTTGTTGTCTTTCCAGTCT 59 9634 9653 405 544147 1062 1081 AATGTTTGTTGTCTTTCCAG 12 9637 9656 406 544148 1095 1114 CGTGATTTCCCAAGTAAAAA 56 9670 9689 407 544149 1160 1179 GTTTTCCGGGATTGCATTGG 33 9735 9754 408 544150 1165 1184 TCTTTGTTTTCCGGGATTGC 54 9740 9759 409 544151 1170 1189 CCAAATCTTTGTTTTCCGGG 64 9745 9764 410 544152 1173 1192 ACACCAAATCTTTGTTTTCC 37 9748 9767 411 544153 1178 1197 AGAAAACACCAAATCTTTGT 32 9753 9772 412 544154 1183 1202 CAAGTAGAAAACACCAAATC 13 9758 9777 413 544155 1188 1207 GATCCCAAGTAGAAAACACC 0 9763 9782 414 544156 1195 1214 GCTTTGTGATCCCAAGTAGA 74 9770 9789 17 544157 1198 1217 TTTGCTTTGTGATCCCAAGT 73 9773 9792 415 544158 1202 1221 TCCTTTTGCTTTGTGATCCC 62 9777 9796 416 544159 1208 1227 GAAGTGTCCTTTTGCTTTGT 30 9783 9802 417 544160 1246 1265 TGCCACCACCAGCCTCCTGA 60 N/A N/A 418 544161 1253 1272 CTCATCATGCCACCACCAGC 73 10225 10244 419 544162 1269 1288 GGTTGTTTTCTCCACACTCA 76 10241 10260 18 544163 1276 1295 CCATTTAGGTTGTTTTCTCC 25 10248 10267 420 544164 1283 1302 ATATTTACCATTTAGGTTGT 25 10255 10274 421 544165 1294 1313 CTTGGTTTGTTATATTTACC 63 10266 10285 422 544166 1353 1372 ACCTTCCATTTTGAGACTTC 75 10325 10344 19 544167 1363 1382 ATAGAGTATAACCTTCCATT 71 10335 10354 423 544168 1367 1386 TTTTATAGAGTATAACCTTC 37 10339 10358 424 544169 1374 1393 TGGTTGATTTTATAGAGTAT 37 10346 10365 425 544170 1378 1397 ATTTTGGTTGATTTTATAGA 3 10350 10369 426 544171 1383 1402 TCAACATTTTGGTTGATTTT 16 10355 10374 427 544172 1390 1409 GGATGGATCAACATTTTGGT 51 10362 10381 428 544173 1393 1412 GTTGGATGGATCAACATTTT 62 10365 10384 429 544174 1396 1415 TCTGTTGGATGGATCAACAT 5 10368 10387 430 544175 1401 1420 CTGAATCTGTTGGATGGATC 55 10373 10392 431 544176 1407 1426 AGCTTTCTGAATCTGTTGGA 65 10379 10398 432 544177 1414 1433 CATTCAAAGCTTTCTGAATC 21 10386 10405 433 544178 1417 1436 GTTCATTCAAAGCTTTCTGA 66 10389 10408 434 544179 1420 1439 TCAGTTCATTCAAAGCTTTC 6 10392 10411 435 544180 1423 1442 GCCTCAGTTCATTCAAAGCT 68 10395 10414 436 544181 1427 1446 ATTTGCCTCAGTTCATTCAA 53 10399 10418 437 544182 1431 1450 TTAAATTTGCCTCAGTTCAT 40 10403 10422 438 544183 1436 1455 GCCTTTTAAATTTGCCTCAG 70 10408 10427 439 544184 1498 1517 AGGATTTAATACCAGATTAT 38 10470 10489 440 544185 1502 1521 CTTAAGGATTTAATACCAGA 56 10474 10493 441 544186 1505 1524 TCTCTTAAGGATTTAATACC 33 10477 10496 442 544187 1546 1565 GACAGTGACTTTAAGATAAA 35 10518 10537 443 544188 1572 1591 TGTGATTGTATGTTTAATCT 48 10544 10563 444 544189 1578 1597 AGGTTATGTGATTGTATGTT 48 10550 10569 445 544190 1583 1602 CTTTAAGGTTATGTGATTGT 48 10555 10574 446 544191 1589 1608 GGTATTCTTTAAGGTTATGT 62 10561 10580 447 544192 1656 1675 ATTGATTCCCACATCACAAA 47 10628 10647 448 544193 1661 1680 CTAAAATTGATTCCCACATC 67 10633 10652 449 544194 1665 1684 CCATCTAAAATTGATTCCCA 63 10637 10656 450 544195 1771 1790 TTGTGATATTAGCTCATATG 59 10743 10762 451 544196 1794 1813 ACTAGTTTTTTAAACTGGGA 28 10766 10785 452 544197 1820 1839 GTCAAGTTTAGAGTTTTAAC 44 10792 10811 453 544198 1826 1845 TATTTAGTCAAGTTTAGAGT 14 10798 10817 454 544199 1907 1926 TACACATACTCTGTGCTGAC 82 10879 10898 20 544200 1913 1932 GATTTTTACACATACTCTGT 57 10885 10904 455 544201 2008 2027 CTGCTTCATTAGGTTTCATA 61 10980 10999 456

TABLE 128 Inhibition of ANGPTL3 mRNA by 5-10-5 MOE gapmers targeting SEQ ID NO: 1 and 2 SEQ ID SEQ ID SEQ ID SEQ ID NO: 1 NO: NO: 2 NO: 2 ISIS Start 1 Stop % Start Stop SEQ ID NO Site Site Sequence inhibition Site Site NO 544127 765 784 CAGCAGGAATGCCATCATGT 0 N/A N/A 457 544128 819 838 TGATGGCATACATGCCACTT 13 7404 7423 458 544129 828 847 TGCTGGGTCTGATGGCATAC 49 7413 7432 459 544130 832 851 GAGTTGCTGGGTCTGATGGC 27 7417 7436 460 544131 841 860 AAAACTTGAGAGTTGCTGGG 0 7426 7445 461 544132 848 867 GACATGAAAAACTTGAGAGT 0 7433 7452 462 544133 859 878 ACATCACAGTAGACATGAAA 18 7444 7463 463 233717 889 908 TGAATTAATGTCCATGGACT 55 7876 7895 14 544134 915 934 AGTTTTGTGATCCATCTATT 68 7902 7921 464 544135 918 937 TGAAGTTTTGTGATCCATCT 77 7905 7924 465 544136 926 945 CGTTTCATTGAAGTTTTGTG 60 7913 7932 466 544137 946 965 CCATATTTGTAGTTCTCCCA 64 7933 7952 467 544138 949 968 AAACCATATTTGTAGTTCTC 45 7936 7955 468 544139 970 989 AATTCTCCATCAAGCCTCCC 70 N/A N/A 469 233722 991 1010 ATCTTCTCTAGGCCCAACCA 96 9566 9585 470 544432 997 1016 GAGTATATCTTCTCTAGGCC 69 9572 9591 471 544140 1002 1021 CTATGGAGTATATCTTCTCT 37 9577 9596 472 544141 1008 1027 GCTTCACTATGGAGTATATC 65 9583 9602 473 544142 1013 1032 AGATTGCTTCACTATGGAGT 55 9588 9607 474 544143 1046 1065 CCAGTCTTCCAACTCAATTC 31 9621 9640 475 544144 1052 1071 GTCTTTCCAGTCTTCCAACT 72 9627 9646 476 544145 1055 1074 GTTGTCTTTCCAGTCTTCCA 86 9630 9649 16 544146 1059 1078 GTTTGTTGTCTTTCCAGTCT 66 9634 9653 477 544147 1062 1081 AATGTTTGTTGTCTTTCCAG 21 9637 9656 478 544148 1095 1114 CGTGATTTCCCAAGTAAAAA 63 9670 9689 479 544149 1160 1179 GTTTTCCGGGATTGCATTGG 32 9735 9754 480 544150 1165 1184 TCTTTGTTTTCCGGGATTGC 48 9740 9759 481 544151 1170 1189 CCAAATCTTTGTTTTCCGGG 72 9745 9764 482 544152 1173 1192 ACACCAAATCTTTGTTTTCC 39 9748 9767 483 544153 1178 1197 AGAAAACACCAAATCTTTGT 39 9753 9772 484 544154 1183 1202 CAAGTAGAAAACACCAAATC 22 9758 9777 485 544155 1188 1207 GATCCCAAGTAGAAAACACC 5 9763 9782 486 544156 1195 1214 GCTTTGTGATCCCAAGTAGA 79 9770 9789 17 544157 1198 1217 TTTGCTTTGTGATCCCAAGT 80 9773 9792 487 544158 1202 1221 TCCTTTTGCTTTGTGATCCC 73 9777 9796 488 544159 1208 1227 GAAGTGTCCTTTTGCTTTGT 33 9783 9802 489 544160 1246 1265 TGCCACCACCAGCCTCCTGA 67 N/A N/A 490 544161 1253 1272 CTCATCATGCCACCACCAGC 79 10225 10244 491 544162 1269 1288 GGTTGTTTTCTCCACACTCA 84 10241 10260 18 544163 1276 1295 CCATTTAGGTTGTTTTCTCC 34 10248 10267 492 544164 1283 1302 ATATTTACCATTTAGGTTGT 17 10255 10274 493 544165 1294 1313 CTTGGTTTGTTATATTTACC 76 10266 10285 494 544166 1353 1372 ACCTTCCATTTTGAGACTTC 79 10325 10344 19 544167 1363 1382 ATAGAGTATAACCTTCCATT 73 10335 10354 495 544168 1367 1386 TTTTATAGAGTATAACCTTC 41 10339 10358 496 544169 1374 1393 TGGTTGATTTTATAGAGTAT 53 10346 10365 497 544170 1378 1397 ATTTTGGTTGATTTTATAGA 28 10350 10369 498 544171 1383 1402 TCAACATTTTGGTTGATTTT 19 10355 10374 499 544172 1390 1409 GGATGGATCAACATTTTGGT 66 10362 10381 500 544173 1393 1412 GTTGGATGGATCAACATTTT 71 10365 10384 501 544174 1396 1415 TCTGTTGGATGGATCAACAT 35 10368 10387 502 544175 1401 1420 CTGAATCTGTTGGATGGATC 68 10373 10392 503 544176 1407 1426 AGCTTTCTGAATCTGTTGGA 70 10379 10398 504 544177 1414 1433 CATTCAAAGCTTTCTGAATC 35 10386 10405 505 544178 1417 1436 GTTCATTCAAAGCTTTCTGA 76 10389 10408 506 544179 1420 1439 TCAGTTCATTCAAAGCTTTC 15 10392 10411 507 544180 1423 1442 GCCTCAGTTCATTCAAAGCT 68 10395 10414 508 544181 1427 1446 ATTTGCCTCAGTTCATTCAA 67 10399 10418 509 544182 1431 1450 TTAAATTTGCCTCAGTTCAT 51 10403 10422 510 544183 1436 1455 GCCTTTTAAATTTGCCTCAG 80 10408 10427 511 544184 1498 1517 AGGATTTAATACCAGATTAT 54 10470 10489 512 544185 1502 1521 CTTAAGGATTTAATACCAGA 69 10474 10493 513 544186 1505 1524 TCTCTTAAGGATTTAATACC 58 10477 10496 514 544187 1546 1565 GACAGTGACTTTAAGATAAA 34 10518 10537 515 544188 1572 1591 TGTGATTGTATGTTTAATCT 47 10544 10563 516 544189 1578 1597 AGGTTATGTGATTGTATGTT 68 10550 10569 517 544190 1583 1602 CTTTAAGGTTATGTGATTGT 62 10555 10574 518 544191 1589 1608 GGTATTCTTTAAGGTTATGT 66 10561 10580 519 544192 1656 1675 ATTGATTCCCACATCACAAA 50 10628 10647 520 544193 1661 1680 CTAAAATTGATTCCCACATC 73 10633 10652 521 544194 1665 1684 CCATCTAAAATTGATTCCCA 73 10637 10656 522 544195 1771 1790 TTGTGATATTAGCTCATATG 57 10743 10762 523 544196 1794 1813 ACTAGTTTTTTAAACTGGGA 21 10766 10785 524 544197 1820 1839 GTCAAGTTTAGAGTTTTAAC 53 10792 10811 525 544198 1826 1845 TATTTAGTCAAGTTTAGAGT 11 10798 10817 526 544199 1907 1926 TACACATACTCTGTGCTGAC 84 10879 10898 20 544200 1913 1932 GATTTTTACACATACTCTGT 53 10885 10904 527 544201 2008 2027 CTGCTTCATTAGGTTTCATA 67 10980 10999 528

TABLE 129 Inhibition of ANGPTL3 mRNA by 5-10-5 MOE gapmers targeting SEQ ID NO: 1 and 2 SEQ ID SEQ ID SEQ ID SEQ ID NO: 1 NO: NO: 2 NO: 2 ISIS Start 1 Stop % Start Stop SEQ ID NO Site Site Sequence inhibition Site Site NO 544127 765 784 CAGCAGGAATGCCATCATGT 18 N/A N/A 529 544128 819 838 TGATGGCATACATGCCACTT 0 7404 7423 530 544129 828 847 TGCTGGGTCTGATGGCATAC 48 7413 7432 531 544130 832 851 GAGTTGCTGGGTCTGATGGC 14 7417 7436 532 544131 841 860 AAAACTTGAGAGTTGCTGGG 5 7426 7445 533 544132 848 867 GACATGAAAAACTTGAGAGT 0 7433 7452 534 544133 859 878 ACATCACAGTAGACATGAAA 28 7444 7463 535 233717 889 908 TGAATTAATGTCCATGGACT 51 7876 7895 14 544134 915 934 AGTTTTGTGATCCATCTATT 36 7902 7921 536 544135 918 937 TGAAGTTTTGTGATCCATCT 61 7905 7924 537 544136 926 945 CGTTTCATTGAAGTTTTGTG 54 7913 7932 538 544137 946 965 CCATATTTGTAGTTCTCCCA 67 7933 7952 539 544138 949 968 AAACCATATTTGTAGTTCTC 39 7936 7955 540 544139 970 989 AATTCTCCATCAAGCCTCCC 77 N/A N/A 541 233722 991 1010 ATCTTCTCTAGGCCCAACCA 95 9566 9585 542 544432 997 1016 GAGTATATCTTCTCTAGGCC 86 9572 9591 543 544140 1002 1021 CTATGGAGTATATCTTCTCT 57 9577 9596 544 544141 1008 1027 GCTTCACTATGGAGTATATC 52 9583 9602 545 544142 1013 1032 AGATTGCTTCACTATGGAGT 40 9588 9607 546 544143 1046 1065 CCAGTCTTCCAACTCAATTC 32 9621 9640 547 544144 1052 1071 GTCTTTCCAGTCTTCCAACT 53 9627 9646 548 544145 1055 1074 GTTGTCTTTCCAGTCTTCCA 80 9630 9649 16 544146 1059 1078 GTTTGTTGTCTTTCCAGTCT 59 9634 9653 549 544147 1062 1081 AATGTTTGTTGTCTTTCCAG 42 9637 9656 550 544148 1095 1114 CGTGATTTCCCAAGTAAAAA 76 9670 9689 551 544149 1160 1179 GTTTTCCGGGATTGCATTGG 29 9735 9754 552 544150 1165 1184 TCTTTGTTTTCCGGGATTGC 50 9740 9759 553 544151 1170 1189 CCAAATCTTTGTTTTCCGGG 56 9745 9764 554 544152 1173 1192 ACACCAAATCTTTGTTTTCC 26 9748 9767 555 544153 1178 1197 AGAAAACACCAAATCTTTGT 22 9753 9772 556 544154 1183 1202 CAAGTAGAAAACACCAAATC 29 9758 9777 557 544155 1188 1207 GATCCCAAGTAGAAAACACC 16 9763 9782 558 544156 1195 1214 GCTTTGTGATCCCAAGTAGA 71 9770 9789 17 544157 1198 1217 TTTGCTTTGTGATCCCAAGT 55 9773 9792 559 544158 1202 1221 TCCTTTTGCTTTGTGATCCC 51 9777 9796 560 544159 1208 1227 GAAGTGTCCTTTTGCTTTGT 8 9783 9802 561 544160 1246 1265 TGCCACCACCAGCCTCCTGA 68 N/A N/A 562 544161 1253 1272 CTCATCATGCCACCACCAGC 48 10225 10244 563 544162 1269 1288 GGTTGTTTTCTCCACACTCA 74 10241 10260 18 544163 1276 1295 CCATTTAGGTTGTTTTCTCC 33 10248 10267 564 544164 1283 1302 ATATTTACCATTTAGGTTGT 0 10255 10274 565 544165 1294 1313 CTTGGTTTGTTATATTTACC 52 10266 10285 566 544166 1353 1372 ACCTTCCATTTTGAGACTTC 69 10325 10344 19 544167 1363 1382 ATAGAGTATAACCTTCCATT 72 10335 10354 567 544168 1367 1386 TTTTATAGAGTATAACCTTC 27 10339 10358 568 544169 1374 1393 TGGTTGATTTTATAGAGTAT 39 10346 10365 569 544170 1378 1397 ATTTTGGTTGATTTTATAGA 7 10350 10369 570 544171 1383 1402 TCAACATTTTGGTTGATTTT 0 10355 10374 571 544172 1390 1409 GGATGGATCAACATTTTGGT 48 10362 10381 572 544173 1393 1412 GTTGGATGGATCAACATTTT 51 10365 10384 573 544174 1396 1415 TCTGTTGGATGGATCAACAT 46 10368 10387 574 544175 1401 1420 CTGAATCTGTTGGATGGATC 58 10373 10392 575 544176 1407 1426 AGCTTTCTGAATCTGTTGGA 57 10379 10398 576 544177 1414 1433 CATTCAAAGCTTTCTGAATC 0 10386 10405 577 544178 1417 1436 GTTCATTCAAAGCTTTCTGA 62 10389 10408 578 544179 1420 1439 TCAGTTCATTCAAAGCTTTC 21 10392 10411 579 544180 1423 1442 GCCTCAGTTCATTCAAAGCT 73 10395 10414 580 544181 1427 1446 ATTTGCCTCAGTTCATTCAA 46 10399 10418 581 544182 1431 1450 TTAAATTTGCCTCAGTTCAT 52 10403 10422 582 544183 1436 1455 GCCTTTTAAATTTGCCTCAG 66 10408 10427 583 544184 1498 1517 AGGATTTAATACCAGATTAT 31 10470 10489 584 544185 1502 1521 CTTAAGGATTTAATACCAGA 49 10474 10493 585 544186 1505 1524 TCTCTTAAGGATTTAATACC 49 10477 10496 586 544187 1546 1565 GACAGTGACTTTAAGATAAA 27 10518 10537 587 544188 1572 1591 TGTGATTGTATGTTTAATCT 30 10544 10563 588 544189 1578 1597 AGGTTATGTGATTGTATGTT 35 10550 10569 589 544190 1583 1602 CTTTAAGGTTATGTGATTGT 50 10555 10574 590 544191 1589 1608 GGTATTCTTTAAGGTTATGT 54 10561 10580 591 544192 1656 1675 ATTGATTCCCACATCACAAA 47 10628 10647 592 544193 1661 1680 CTAAAATTGATTCCCACATC 69 10633 10652 593 544194 1665 1684 CCATCTAAAATTGATTCCCA 74 10637 10656 594 544195 1771 1790 TTGTGATATTAGCTCATATG 54 10743 10762 595 544196 1794 1813 ACTAGTTTTTTAAACTGGGA 27 10766 10785 596 544197 1820 1839 GTCAAGTTTAGAGTTTTAAC 18 10792 10811 597 544198 1826 1845 TATTTAGTCAAGTTTAGAGT 12 10798 10817 598 544199 1907 1926 TACACATACTCTGTGCTGAC 83 10879 10898 20 544200 1913 1932 GATTTTTACACATACTCTGT 58 10885 10904 599 544201 2008 2027 CTGCTTCATTAGGTTTCATA 62 10980 10999 600

TABLE 130 Inhibition of ANGPTL3 mRNA by 5-10-5 MOE gapmers targeting SEQ ID NO: 1 and 2 SEQ ID SEQ ID SEQ ID SEQ ID NO: 1 NO: NO: 2 NO: 2 ISIS Start 1 Stop % Start Stop SEQ ID NO Site Site Sequence inhibition Site Site NO 337520 N/A N/A CAGTGTTATTCAGATTGTAC 64 6517 6536 601 337521 N/A N/A AGTGTCTTACCATCATGTTT 40 6776 6795 602 337525 N/A N/A CACCAGCCTCCTAAAGGAGA 39 10212 10231 603 544292 N/A N/A GAGGAGGTGAAGTCAGTGAG 35 4815 4834 604 544293 N/A N/A TAGAGTAGAGGAGGTGAAGT 23 4822 4841 605 544294 N/A N/A TGTTTGATGTGTTTGAATAC 19 4863 4882 606 544295 N/A N/A GAAACAACAAGGGCAAAGGC 23 4898 4917 607 544296 N/A N/A TGTTTGATAACGACCCTAAG 43 4974 4993 608 544297 N/A N/A TTTTTGGTTAAGTGACCTTG 48 5016 5035 609 544298 N/A N/A GTAGAAGTTTTCAGGGATGG 23 5052 5071 610 544299 N/A N/A AGGAAGTAGAAGTTTTCAGG 5 5057 5076 611 544300 N/A N/A AGGTGAGTGTGCAGGAGAAA 11 5085 5104 612 544301 N/A N/A TTAAATAAAGGTGAGTGTGC 14 5093 5112 613 544302 N/A N/A AGTGCAGGAATAGAAGAGAT 35 5136 5155 614 544303 N/A N/A CATTTTAGTGCAGGAATAGA 21 5142 5161 615 544306 N/A N/A CTATATTCTGGAGTATATAC 39 5216 5235 616 544307 N/A N/A CAGTATTCTATATTCTGGAG 72 5223 5242 617 544308 N/A N/A GTGCCATACAGTATTCTATA 50 5231 5250 618 544309 N/A N/A CTGTGTGAATATGACATTAC 52 5281 5300 619 544310 N/A N/A TGAGGCACACTATTTCTAGT 47 5333 5352 620 544311 N/A N/A GACCTTTAATTATGAGGCAC 67 5345 5364 621 544312 N/A N/A GAATGTTGACCTTTAATTAT 23 5352 5371 622 544313 N/A N/A TTGTTGAATGTTGACCTTTA 69 5357 5376 623 544314 N/A N/A TCTACTAAGTAACTATGTGA 37 5915 5934 624 544315 N/A N/A CTCTTTTCTACTAAGTAACT 31 5921 5940 625 544316 N/A N/A AAGGATCTATTGTAAAGTTT 24 5956 5975 626 544317 N/A N/A CTAGGACCTTATTTAAAAGG 24 5972 5991 627 544318 N/A N/A ATTTCCTAGGACCTTATTTA 8 5977 5996 628 544319 N/A N/A TTGACAGTAAGAAAAGCAGA 28 6051 6070 629 544320 N/A N/A TTCTCATTGACAGTAAGAAA 56 6057 6076 630 544321 N/A N/A AGTTTTTCTCATTGACAGTA 50 6062 6081 631 544322 N/A N/A ATTGAATGATAGTTTTTCTC 42 6072 6091 632 544323 N/A N/A TTGGGTTTGCAATTTATTGA 36 6087 6106 633 544324 N/A N/A AGTGTGTTGGGTTTGCAATT 25 6093 6112 634 544325 N/A N/A TATTTAAGTGTGTTGGGTTT 27 6099 6118 635 544326 N/A N/A ATATATTCAGTAGTTTATCG 25 6145 6164 636 544327 N/A N/A AGATGTTGGCAGGTTGGCAA 51 6184 6203 637 544328 N/A N/A TCTGTAGATGTTGGCAGGTT 48 6189 6208 638 544329 N/A N/A TTGATAATTTTTGACCTGTA 34 6215 6234 639 544330 N/A N/A GGCTTTCTTGATAATTTGAT 52 6230 6249 640 544331 N/A N/A GTCTTACTGATCTTCAGACC 27 6282 6301 641 544332 N/A N/A TTTAGGTCTTACTGATCTTC 14 6287 6306 642 544333 N/A N/A TCAGTTTTAGGTCTTACTGA 28 6292 6311 643 544334 N/A N/A TGATATTCTGTTCAGATTTT 44 6326 6345 644 544335 N/A N/A TAGAGACTGCTTTGCTTAGA 31 6388 6407 645 544336 N/A N/A AGGCCAAAAGTAGAGACTGC 29 6398 6417 646 544337 N/A N/A GGCAAAAAAGCAGACATTGG 38 6433 6452 647 544338 N/A N/A AATCAGGGACATTATTTAAT 13 6473 6492 648 544339 N/A N/A TATTTAATCAGGGACATTAT 28 6478 6497 649 544340 N/A N/A CTCAAAATATTTAATCAGGG 27 6485 6504 650 544341 N/A N/A TACCTGTTCTCAAAATATTT 18 6493 6512 651 544342 N/A N/A GTACAGATTACCTGTTCTCA 68 6501 6520 652 544343 N/A N/A GGTGTTTGATATTTAGATAA 25 6538 6557 653 544344 N/A N/A TTGTCTTTCAGTTCATAATG 29 6565 6584 654 544345 N/A N/A ACAGTTTGTCTTTCAGTTCA 23 6570 6589 655 544346 N/A N/A TCTGAGCTGATAAAAGAATA 15 6657 6676 656 544347 N/A N/A CCCACCAAAGTGTCTTACCA 49 6784 6803 657 544348 N/A N/A CTTCAAGAAGGAAACCCACC 39 6798 6817 658 544349 N/A N/A AATAGCTTCAAGAAGGAAAC 12 6803 6822 659 544350 N/A N/A ACAAGTCCTAAGAATAGGGA 25 6833 6852 660 544351 N/A N/A GTCTAGAACAAGTCCTAAGA 53 6840 6859 661 544352 N/A N/A TCTAATAATCAAGTCCATAT 33 6972 6991 662 544353 N/A N/A ACCTTCTATATTATCTAATA 19 6985 7004 663 544354 N/A N/A GCATGTATCTCTTAAACAGG 50 7060 7079 664 544355 N/A N/A TTTCAGCATGTATCTCTTAA 79 7065 7084 21 544356 N/A N/A GTCCAGTGACCTTTAACTCC 69 7092 7111 665 544357 N/A N/A TCTTACCAAACTATTTTCTT 28 7166 7185 666 544358 N/A N/A GTAATGTTTATGTTAAAGCA 17 7226 7245 667 544359 N/A N/A TTGTGGCAAATGTAGCATTT 52 7251 7270 668 544360 N/A N/A GAGATTTCACTTGACATTTT 30 7277 7296 669 544361 N/A N/A GGAGCTTGAGATTTCACTTG 30 7284 7303 670 544362 N/A N/A CATCAGATTTAGTAATAGGA 0 7315 7334 671 544363 N/A N/A GTTATTACATCAGATTTAGT 6 7322 7341 672 544365 N/A N/A CAGCAGGAATGCCTAGAATC 32 7350 7369 673 544366 N/A N/A CTCCTTAGACAGGTTTTACC 31 7471 7490 674 544367 N/A N/A GTCTATTCTCCTTAGACAGG 23 7478 7497 675 544368 N/A N/A ACCAGGTTAATCTTCCTAAT 71 7526 7545 22 544369 N/A N/A ATGAATGATTGAATGTAGTC 26 7977 7996 676 544370 N/A N/A ATATGAAGGCTGAGACTGCT 58 8072 8091 677 544371 N/A N/A ATAAATTATATGAAGGCTGA 7 8079 8098 678 544372 N/A N/A ATATTTAAGAACAGACATGT 12 8175 8194 679 544373 N/A N/A AGTTATGATCATTGTAAGCC 60 8217 8236 23 544374 N/A N/A ATTTGTAACAGTTACTACTT 51 8276 8295 680 544375 N/A N/A CACAGCTTATTTGTAACAGT 70 8284 8303 681 544376 N/A N/A GGAGTGGTTCTTTTCACAGC 71 8298 8317 24 544377 N/A N/A GTGACTAATGCTAGGAGTGG 34 8311 8330 682 544378 N/A N/A GAATAGAGTGACTAATGCTA 45 8318 8337 683 544379 N/A N/A ATGAGAGAATAGAGTGACTA 58 8324 8343 684 544380 N/A N/A TGGTCCTTTTAACTTCCAAT 70 8365 8384 25 544381 N/A N/A TATACTGTATGTCTGAGTTT 66 8387 8406 685 544382 N/A N/A AACTAATTCATTATAAGCCA 67 8450 8469 686 544383 N/A N/A GCATTGAGTTAACTAATTCA 64 8460 8479 26 544385 N/A N/A TTTGGATTTTAAACATCTGT 61 8528 8547 687 544386 N/A N/A TGTATGTGCTTTTTGGATTT 37 8539 8558 688 544387 N/A N/A CATGGATTTTTGTATGTGCT 62 8549 8568 689 544388 N/A N/A TCATTCATGGATTTTTGTAT 34 8554 8573 690 544389 N/A N/A ACTTAGACATCATTCATGGA 55 8563 8582 691 544390 N/A N/A GTGAGTACTTAGACATCATT 66 8569 8588 692 544391 N/A N/A TTTATAAGTGAGTACTTAGA 36 8576 8595 693 544392 N/A N/A GTCTTCTACTTTATAAGTGA 65 8585 8604 694 544393 N/A N/A ATGAATGTCTTCTACTTTAT 34 8591 8610 695 544394 N/A N/A CAAATAGTACTGAGCATTTA 30 8627 8646 696 544395 N/A N/A TTAGAAGATTTGGAGCTACA 54 8718 8737 697 544396 N/A N/A TCACTATTAGAAGATTTGGA 37 8724 8743 698 544397 N/A N/A GGGTTACACTCACTATTAGA 36 8733 8752 699 544398 N/A N/A ACTTACCTGTCAGCCTTTTA 54 8758 8777 700 544399 N/A N/A CTTACCAGAATTAAGTGAGT 26 8785 8804 701 544400 N/A N/A AATACAAGTACAAATGGGTT 22 8810 8829 702 544401 N/A N/A CTGGTAAATACAAGTACAAA 55 8816 8835 703 544402 N/A N/A GGATTGCTGGTAAATACAAG 40 8822 8841 704 544403 N/A N/A TCATTTTAAGGATTGCTGGT 62 8831 8850 705 544404 N/A N/A AGTTAGTAGGAAGCTTCATT 56 8846 8865 706 544405 N/A N/A GCTATTGAGTTAGTAGGAAG 67 8853 8872 707 544407 N/A N/A AGCATGGTTCTTAATAACTT 67 9012 9031 708 544408 N/A N/A CTTTGTAGAAAAAGACAGGA 27 9062 9081 709 544409 N/A N/A ACCTGGCCTTTGGTATTTGC 49 9096 9115 710 544410 N/A N/A CATCCATATACAGTCAAGAG 80 9174 9193 27 544411 N/A N/A AGTCTTTATATGGATAAACT 15 9215 9234 711 544412 N/A N/A CGTCATTGGTAGAGGAATAT 51 9240 9259 712 544413 N/A N/A GATTATCCTTTCTATAATGC 48 9321 9340 713 544414 N/A N/A GTCTTGAATCCCTTGATCAT 40 9436 9455 714 544415 N/A N/A GGTGCAACTAATTGAGTTGT 27 9459 9478 715 544416 N/A N/A GTGTTTTTTATTGGTGCAAC 31 9471 9490 716 544417 N/A N/A ATTCTCCTGAAAAGAAAAGT 24 9544 9563 717 544418 N/A N/A ATGCCACCACCAGCCTCCTA 73 10219 10238 718 544419 N/A N/A ATATCCTTTAACAAATGGGT 62 11540 11559 719 544420 N/A N/A GCACTATATCCTTTAACAAA 50 11545 11564 720 544421 N/A N/A ACTTGGGCACTATATCCTTT 68 11551 11570 721 544422 N/A N/A GAAACATGTCCTATGAGAGT 32 11918 11937 722 544424 N/A N/A TTGAGCACTTTAAGCAAAGT 7 12070 12089 723 544425 N/A N/A GGAATTTGAGCACTTTAAGC 34 12075 12094 724 544426 N/A N/A TAGATTAGACAACTGTGAGT 52 12101 12120 725 544427 N/A N/A AAAATGAAGGTCAAGTTTGA 17 12197 12216 726 544428 N/A N/A GTGAAAGCAAAATGAAGGTC 33 12205 12224 727 544429 N/A N/A GTATTGTGAAAGCAAAATGA 39 12210 12229 728 544430 N/A N/A TGGAGAGTATAGTATTGTGA 35 12221 12240 729 544438 N/A N/A AGGAATAGAAGAGATAAATA 10 5131 5150 730 544439 N/A N/A TGGAGTATATACAAATAATG 30 5208 5227 731 544440 N/A N/A TGTTTACATTGTAGATTAAT 15 5381 5400 732 544441 N/A N/A CAGAATATATAATATCTTGC 57 6035 6054 733 544442 N/A N/A TGCAATTTATTGAATGATAG 31 6080 6099 734 544443 N/A N/A CATAATACATAATTTGAACC 0 6251 6270 735 544444 N/A N/A ATAATTTTCAGTTTTAGGTC 0 6299 6318 736 544445 N/A N/A TTTCAGTAATGTTTATGTTA 9 7231 7250 737 544446 N/A N/A AATGCCTAGAATCAATAAAA 36 7343 7362 738 544447 N/A N/A GTAAATATTTGTAGATTAGC 49 8003 8022 739 544448 N/A N/A ACAAATGTGTAATTGTTTGA 25 8101 8120 740 544449 N/A N/A TACTAACAAATGTGTAATTG 35 8106 8125 741 544450 N/A N/A TGATAAGTATATTTAAGAAC 35 8183 8202 742 544451 N/A N/A TTAACTTCCAATTAATTGAT 29 8357 8376 743 544452 N/A N/A TCTGTTATTTTATCTTGCTT 67 8513 8532 744 544453 N/A N/A ATCACAATCCTTTTTATTAA 18 8921 8940 745 544454 N/A N/A AGAGACTTGAGTAATAATAA 25 9137 9156 746 544455 N/A N/A AACAAAATGAAACATGTCCT 59 11926 11945 747 544127 765 784 CAGCAGGAATGCCATCATGT 33 N/A N/A 748 544128 819 838 TGATGGCATACATGCCACTT 13 7404 7423 749 544129 828 847 TGCTGGGTCTGATGGCATAC 53 7413 7432 750 544130 832 851 GAGTTGCTGGGTCTGATGGC 22 7417 7436 751 544131 841 860 AAAACTTGAGAGTTGCTGGG 13 7426 7445 752 544132 848 867 GACATGAAAAACTTGAGAGT 0 7433 7452 753 544133 859 878 ACATCACAGTAGACATGAAA 27 7444 7463 754 233717 889 908 TGAATTAATGTCCATGGACT 58 7876 7895 14 544134 915 934 AGTTTTGTGATCCATCTATT 46 7902 7921 755 544135 918 937 TGAAGTTTTGTGATCCATCT 54 7905 7924 756 544136 926 945 CGTTTCATTGAAGTTTTGTG 40 7913 7932 757 544137 946 965 CCATATTTGTAGTTCTCCCA 45 7933 7952 758 544138 949 968 AAACCATATTTGTAGTTCTC 41 7936 7955 759 544139 970 989 AATTCTCCATCAAGCCTCCC 43 N/A N/A 760 233722 991 1010 ATCTTCTCTAGGCCCAACCA 65 9566 9585 761 544432 997 1016 GAGTATATCTTCTCTAGGCC 40 9572 9591 762 544140 1002 1021 CTATGGAGTATATCTTCTCT 28 9577 9596 763 544141 1008 1027 GCTTCACTATGGAGTATATC 55 9583 9602 764 544142 1013 1032 AGATTGCTTCACTATGGAGT 47 9588 9607 765 544143 1046 1065 CCAGTCTTCCAACTCAATTC 33 9621 9640 766 544144 1052 1071 GTCTTTCCAGTCTTCCAACT 59 9627 9646 767 544145 1055 1074 GTTGTCTTTCCAGTCTTCCA 77 9630 9649 16 544146 1059 1078 GTTTGTTGTCTTTCCAGTCT 58 9634 9653 768 544147 1062 1081 AATGTTTGTTGTCTTTCCAG 43 9637 9656 769 544148 1095 1114 CGTGATTTCCCAAGTAAAAA 57 9670 9689 770 544149 1160 1179 GTTTTCCGGGATTGCATTGG 44 9735 9754 771 544150 1165 1184 TCTTTGTTTTCCGGGATTGC 53 9740 9759 772 544151 1170 1189 CCAAATCTTTGTTTTCCGGG 57 9745 9764 773 544152 1173 1192 ACACCAAATCTTTGTTTTCC 44 9748 9767 774 544153 1178 1197 AGAAAACACCAAATCTTTGT 36 9753 9772 775 544154 1183 1202 CAAGTAGAAAACACCAAATC 29 9758 9777 776 544155 1188 1207 GATCCCAAGTAGAAAACACC 29 9763 9782 777 544156 1195 1214 GCTTTGTGATCCCAAGTAGA 71 9770 9789 17 544157 1198 1217 TTTGCTTTGTGATCCCAAGT 66 9773 9792 778 544158 1202 1221 TCCTTTTGCTTTGTGATCCC 53 9777 9796 779 544159 1208 1227 GAAGTGTCCTTTTGCTTTGT 10 9783 9802 780 544160 1246 1265 TGCCACCACCAGCCTCCTGA 65 N/A N/A 781 544161 1253 1272 CTCATCATGCCACCACCAGC 59 10225 10244 782 544162 1269 1288 GGTTGTTTTCTCCACACTCA 74 10241 10260 18 544163 1276 1295 CCATTTAGGTTGTTTTCTCC 38 10248 10267 783 544164 1283 1302 ATATTTACCATTTAGGTTGT 13 10255 10274 784 544165 1294 1313 CTTGGTTTGTTATATTTACC 53 10266 10285 785 544166 1353 1372 ACCTTCCATTTTGAGACTTC 70 10325 10344 19 544167 1363 1382 ATAGAGTATAACCTTCCATT 69 10335 10354 786 544168 1367 1386 TTTTATAGAGTATAACCTTC 34 10339 10358 787 544169 1374 1393 TGGTTGATTTTATAGAGTAT 38 10346 10365 788 544170 1378 1397 ATTTTGGTTGATTTTATAGA 0 10350 10369 789 544171 1383 1402 TCAACATTTTGGTTGATTTT 12 10355 10374 790 544172 1390 1409 GGATGGATCAACATTTTGGT 58 10362 10381 791 544173 1393 1412 GTTGGATGGATCAACATTTT 66 10365 10384 792 544174 1396 1415 TCTGTTGGATGGATCAACAT 49 10368 10387 793 544175 1401 1420 CTGAATCTGTTGGATGGATC 60 10373 10392 794 544176 1407 1426 AGCTTTCTGAATCTGTTGGA 64 10379 10398 795 544177 1414 1433 CATTCAAAGCTTTCTGAATC 21 10386 10405 796 544178 1417 1436 GTTCATTCAAAGCTTTCTGA 60 10389 10408 797 544179 1420 1439 TCAGTTCATTCAAAGCTTTC 18 10392 10411 798 544180 1423 1442 GCCTCAGTTCATTCAAAGCT 72 10395 10414 799 544181 1427 1446 ATTTGCCTCAGTTCATTCAA 51 10399 10418 800 544182 1431 1450 TTAAATTTGCCTCAGTTCAT 48 10403 10422 801 544183 1436 1455 GCCTTTTAAATTTGCCTCAG 70 10408 10427 802 544184 1498 1517 AGGATTTAATACCAGATTAT 44 10470 10489 803 544185 1502 1521 CTTAAGGATTTAATACCAGA 47 10474 10493 804 544186 1505 1524 TCTCTTAAGGATTTAATACC 44 10477 10496 805 544187 1546 1565 GACAGTGACTTTAAGATAAA 38 10518 10537 806 544188 1572 1591 TGTGATTGTATGTTTAATCT 47 10544 10563 807 544189 1578 1597 AGGTTATGTGATTGTATGTT 43 10550 10569 808 544190 1583 1602 CTTTAAGGTTATGTGATTGT 42 10555 10574 809 544191 1589 1608 GGTATTCTTTAAGGTTATGT 60 10561 10580 810 544192 1656 1675 ATTGATTCCCACATCACAAA 46 10628 10647 811 544193 1661 1680 CTAAAATTGATTCCCACATC 65 10633 10652 812 544194 1665 1684 CCATCTAAAATTGATTCCCA 70 10637 10656 813 544195 1771 1790 TTGTGATATTAGCTCATATG 56 10743 10762 814 544196 1794 1813 ACTAGTTTTTTAAACTGGGA 33 10766 10785 815 544197 1820 1839 GTCAAGTTTAGAGTTTTAAC 39 10792 10811 816 544198 1826 1845 TATTTAGTCAAGTTTAGAGT 21 10798 10817 817 544199 1907 1926 TACACATACTCTGTGCTGAC 80 10879 10898 20 544200 1913 1932 GATTTTTACACATACTCTGT 56 10885 10904 818 544201 2008 2027 CTGCTTCATTAGGTTTCATA 65 10980 10999 819

TABLE 131 Inhibition of ANGPTL3 mRNA by 5-10-5 MOE gapmers targeting SEQ ID NO: 1 and 2 SEQ ID SEQ ID SEQ ID SEQ ID NO: 1 NO: NO: 2 NO: 2 ISIS Start 1 Stop % Start Stop SEQ NO Site Site Sequence inhibition Site Site ID NO 337525 N/A N/A CACCAGCCTCCTAAAGGAGA 58 10212 10231 820 544204 N/A N/A GACTTCTTAACTCTATATAT 67 3076 3095 821 544205 N/A N/A CTAGACTTCTTAACTCTATA 61 3079 3098 822 544206 N/A N/A GACCTAGACTTCTTAACTCT 54 3082 3101 823 544207 N/A N/A GGAAGCAGACCTAGACTTCT 58 3089 3108 824 544208 N/A N/A TCTGGAAGCAGACCTAGACT 48 3092 3111 825 544209 N/A N/A TCTTCTGGAAGCAGACCTAG 54 3095 3114 826 544210 N/A N/A CTAATCTTTAGGGATTTAGG 57 11433 11452 827 544211 N/A N/A TGTATCTAATCTTTAGGGAT 53 11438 11457 828 544213 N/A N/A TAACTTGGGCACTATATCCT 74 11553 11572 829 544214 N/A N/A ATTGACAAAGGTAGGTCACC 79 11576 11595 830 544215 N/A N/A ATATGACATGTATATTGGAT 66 11620 11639 831 544216 N/A N/A TTTTGTACTTTTCTGGAACA 61 11704 11723 832 544217 N/A N/A TAGTCTGTGGTCCTGAAAAT 56 11748 11767 833 544218 N/A N/A AGCTTAGTCTGTGGTCCTGA 72 11752 11771 834 544219 N/A N/A GACAGCTTAGTCTGTGGTCC 74 11755 11774 835 544220 N/A N/A GTATTCTGGCCCTAAAAAAA 52 11789 11808 836 544221 N/A N/A ATTTTGGTATTCTGGCCCTA 56 11795 11814 837 544222 N/A N/A GAAATTGTCCAATTTTTGGG 56 N/A N/A 838 544223 N/A N/A TTTGCATTTGAAATTGTCCA 61 11837 11856 839 544224 N/A N/A GGAAGCAACTCATATATTAA 57 11869 11888 840 544225 N/A N/A TATCAGAAAAAGATACCTGA 56 9821 9840 841 544226 N/A N/A ATAATAGCTAATAATGTGGG 59 9875 9894 842 544227 N/A N/A TGCAGATAATAGCTAATAAT 60 9880 9899 843 544228 N/A N/A TGTCATTGCAGATAATAGCT 79 9886 9905 844 544229 N/A N/A TAAAAGTTGTCATTGCAGAT 59 9893 9912 845 544230 N/A N/A CGGATTTTTAAAAGTTGTCA 61 9901 9920 846 544231 N/A N/A GGGATTCGGATTTTTAAAAG 28 9907 9926 847 544232 N/A N/A TTTGGGATTCGGATTTTTAA 44 9910 9929 848 544233 N/A N/A ACGCTTATTTGGGATTCGGA 72 9917 9936 849 544251 N/A N/A TTTAAGAGATTTACAAGTCA 52 2811 2830 850 544252 N/A N/A GACTACCTGTTTTTAAAAGC 48 2851 2870 851 544253 N/A N/A TATGGTGACTACCTGTTTTT 39 2857 2876 852 544254 N/A N/A ACTTTGCTGTATTATAAACT 35 2890 2909 853 544255 N/A N/A ATTGTATTTAACTTTGCTGT 35 2900 2919 854 544256 N/A N/A GAGCAACTAACTTAATAGGT 42 2928 2947 855 544257 N/A N/A GAAATGAGCAACTAACTTAA 32 2933 2952 856 544258 N/A N/A AATCAAAGAAATGAGCAACT 42 2940 2959 857 544259 N/A N/A ACCTTCTTCCACATTGAGTT 44 2977 2996 858 544260 N/A N/A CACGAATGTAACCTTCTTCC 52 2987 3006 859 544261 N/A N/A TTAACTTGCACGAATGTAAC 45 2995 3014 860 544262 N/A N/A TATATATACCAATATTTGCC 43 3063 3082 861 544263 N/A N/A TCTTAACTCTATATATACCA 49 3072 3091 862 544264 N/A N/A CTTTAAGTGAAGTTACTTCT 53 3632 3651 863 544265 N/A N/A TCTACTTACTTTAAGTGAAG 44 3640 3659 864 544266 N/A N/A GAACCCTCTTTATTTTCTAC 46 3655 3674 865 544267 N/A N/A ACATAAACATGAACCCTCTT 50 3665 3684 866 544268 N/A N/A CCACATTGAAAACATAAACA 57 3676 3695 867 544269 N/A N/A GCATGCCTTAGAAATATTTT 23 3707 3726 868 544270 N/A N/A CAATGCAACAAAGTATTTCA 37 3731 3750 869 544271 N/A N/A CTGGAGATTATTTTTCTTGG 61 3768 3787 870 544272 N/A N/A TTCATATATAACATTAGGGA 14 3830 3849 871 544273 N/A N/A TCAGTGTTTTCATATATAAC 32 3838 3857 872 544274 N/A N/A GACATAGTGTTCTAGATTGT 47 3900 3919 873 544275 N/A N/A CAATAGTGTAATGACATAGT 39 3912 3931 874 544276 N/A N/A TTACTTACCTTCAGTAATTT 35 3933 3952 875 544277 N/A N/A ATCTTTTCCATTTACTGTAT 39 4005 4024 876 544278 N/A N/A AGAAAAAGCCCAGCATATTT 23 4037 4056 877 544279 N/A N/A GTATGCTTCTTTCAAATAGC 46 4130 4149 878 544280 N/A N/A CCTTCCCCTTGTATGCTTCT 47 4140 4159 879 544281 N/A N/A CCTGTAACACTATCATAATC 49 4207 4226 880 544282 N/A N/A TGACTTACCTGATTTTCTAT 24 4384 4403 881 544283 N/A N/A GATGGGACATACCATTAAAA 41 4407 4426 882 544284 N/A N/A GTGAAAGATGGGACATACCA 54 4413 4432 883 544285 N/A N/A CCTGTGTGAAAGATGGGACA 27 4418 4437 884 544286 N/A N/A CATTGGCTGCTATGAATTAA 45 4681 4700 885 544287 N/A N/A GATGACATTGGCTGCTATGA 49 4686 4705 886 544288 N/A N/A GAGAAACATGATCTAATTTG 33 4717 4736 887 544289 N/A N/A ATGGAAAGCTATTGTGTGGT 42 4747 4766 888 544290 N/A N/A GTCTAAAGAGCCAATATGAG 39 4771 4790 889 544291 N/A N/A AATCTTGGTCTAAAGAGCCA 65 4778 4797 890 544361 N/A N/A GGAGCTTGAGATTTCACTTG 66 7284 7303 891 544362 N/A N/A CATCAGATTTAGTAATAGGA 61 7315 7334 892 544363 N/A N/A GTTATTACATCAGATTTAGT 63 7322 7341 893 544365 N/A N/A CAGCAGGAATGCCTAGAATC 72 7350 7369 894 544366 N/A N/A CTCCTTAGACAGGTTTTACC 67 7471 7490 895 544367 N/A N/A GTCTATTCTCCTTAGACAGG 59 7478 7497 896 544368 N/A N/A ACCAGGTTAATCTTCCTAAT 79 7526 7545 22 544369 N/A N/A ATGAATGATTGAATGTAGTC 56 7977 7996 897 544370 N/A N/A ATATGAAGGCTGAGACTGCT 73 8072 8091 898 544371 N/A N/A ATAAATTATATGAAGGCTGA 51 8079 8098 899 544372 N/A N/A ATATTTAAGAACAGACATGT 54 8175 8194 900 544373 N/A N/A AGTTATGATCATTGTAAGCC 77 8217 8236 23 544374 N/A N/A ATTTGTAACAGTTACTACTT 69 8276 8295 901 544375 N/A N/A CACAGCTTATTTGTAACAGT 72 8284 8303 902 544376 N/A N/A GGAGTGGTTCTTTTCACAGC 82 8298 8317 24 544377 N/A N/A GTGACTAATGCTAGGAGTGG 54 8311 8330 903 544378 N/A N/A GAATAGAGTGACTAATGCTA 55 8318 8337 904 544379 N/A N/A ATGAGAGAATAGAGTGACTA 66 8324 8343 905 544380 N/A N/A TGGTCCTTTTAACTTCCAAT 79 8365 8384 25 544381 N/A N/A TATACTGTATGTCTGAGTTT 72 8387 8406 906 544382 N/A N/A AACTAATTCATTATAAGCCA 56 8450 8469 907 544383 N/A N/A GCATTGAGTTAACTAATTCA 78 8460 8479 26 544385 N/A N/A TTTGGATTTTAAACATCTGT 73 8528 8547 908 544386 N/A N/A TGTATGTGCTTTTTGGATTT 57 8539 8558 909 544387 N/A N/A CATGGATTTTTGTATGTGCT 64 8549 8568 910 544388 N/A N/A TCATTCATGGATTTTTGTAT 53 8554 8573 911 544389 N/A N/A ACTTAGACATCATTCATGGA 66 8563 8582 912 544390 N/A N/A GTGAGTACTTAGACATCATT 74 8569 8588 913 544391 N/A N/A TTTATAAGTGAGTACTTAGA 32 8576 8595 914 544392 N/A N/A GTCTTCTACTTTATAAGTGA 63 8585 8604 915 544393 N/A N/A ATGAATGTCTTCTACTTTAT 68 8591 8610 916 544394 N/A N/A CAAATAGTACTGAGCATTTA 53 8627 8646 917 544395 N/A N/A TTAGAAGATTTGGAGCTACA 55 8718 8737 918 544396 N/A N/A TCACTATTAGAAGATTTGGA 60 8724 8743 919 544397 N/A N/A GGGTTACACTCACTATTAGA 52 8733 8752 920 544398 N/A N/A ACTTACCTGTCAGCCTTTTA 61 8758 8777 921 544399 N/A N/A CTTACCAGAATTAAGTGAGT 43 8785 8804 922 544400 N/A N/A AATACAAGTACAAATGGGTT 29 8810 8829 923 544401 N/A N/A CTGGTAAATACAAGTACAAA 76 8816 8835 924 544402 N/A N/A GGATTGCTGGTAAATACAAG 59 8822 8841 925 544403 N/A N/A TCATTTTAAGGATTGCTGGT 63 8831 8850 926 544404 N/A N/A AGTTAGTAGGAAGCTTCATT 54 8846 8865 927 544405 N/A N/A GCTATTGAGTTAGTAGGAAG 63 8853 8872 928 544407 N/A N/A AGCATGGTTCTTAATAACTT 69 9012 9031 929 544408 N/A N/A CTTTGTAGAAAAAGACAGGA 45 9062 9081 930 544409 N/A N/A ACCTGGCCTTTGGTATTTGC 66 9096 9115 931 544410 N/A N/A CATCCATATACAGTCAAGAG 78 9174 9193 27 544411 N/A N/A AGTCTTTATATGGATAAACT 46 9215 9234 932 544412 N/A N/A CGTCATTGGTAGAGGAATAT 45 9240 9259 933 544413 N/A N/A GATTATCCTTTCTATAATGC 45 9321 9340 934 544414 N/A N/A GTCTTGAATCCCTTGATCAT 61 9436 9455 935 544415 N/A N/A GGTGCAACTAATTGAGTTGT 49 9459 9478 936 544416 N/A N/A GTGTTTTTTATTGGTGCAAC 46 9471 9490 937 544417 N/A N/A ATTCTCCTGAAAAGAAAAGT 50 9544 9563 938 544418 N/A N/A ATGCCACCACCAGCCTCCTA 73 10219 10238 939 544419 N/A N/A ATATCCTTTAACAAATGGGT 68 11540 11559 940 544420 N/A N/A GCACTATATCCTTTAACAAA 74 11545 11564 941 544421 N/A N/A ACTTGGGCACTATATCCTTT 68 11551 11570 942 544422 N/A N/A GAAACATGTCCTATGAGAGT 56 11918 11937 943 544424 N/A N/A TTGAGCACTTTAAGCAAAGT 15 12070 12089 944 544425 N/A N/A GGAATTTGAGCACTTTAAGC 35 12075 12094 945 544426 N/A N/A TAGATTAGACAACTGTGAGT 54 12101 12120 946 544427 N/A N/A AAAATGAAGGTCAAGTTTGA 45 12197 12216 947 544428 N/A N/A GTGAAAGCAAAATGAAGGTC 55 12205 12224 948 544429 N/A N/A GTATTGTGAAAGCAAAATGA 54 12210 12229 949 544430 N/A N/A TGGAGAGTATAGTATTGTGA 53 12221 12240 950 544433 N/A N/A GAGATTTACAAGTCAAAAAT 41 2806 2825 951 544434 N/A N/A ATTTAACTTTGCTGTATTAT 29 2895 2914 952 544435 N/A N/A ATCAATGCTAAATGAAATCA 34 2955 2974 953 544436 N/A N/A TATTTTCTGGAGATTATTTT 24 3774 3793 954 544437 N/A N/A AAAATGAATATTGGCAATTC 34 4159 4178 955 544446 N/A N/A AATGCCTAGAATCAATAAAA 50 7343 7362 956 544447 N/A N/A GTAAATATTTGTAGATTAGC 38 8003 8022 957 544448 N/A N/A ACAAATGTGTAATTGTTTGA 43 8101 8120 958 544449 N/A N/A TACTAACAAATGTGTAATTG 59 8106 8125 959 544450 N/A N/A TGATAAGTATATTTAAGAAC 45 8183 8202 960 544451 N/A N/A TTAACTTCCAATTAATTGAT 55 8357 8376 961 544452 N/A N/A TCTGTTATTTTATCTTGCTT 67 8513 8532 962 544453 N/A N/A ATCACAATCCTTTTTATTAA 39 8921 8940 963 544454 N/A N/A AGAGACTTGAGTAATAATAA 43 9137 9156 964 544455 N/A N/A AACAAAATGAAACATGTCCT 47 11926 11945 965 544059 23 42 GATTTTCAATTTCAAGCAAC 74 3127 3146 966 337459 49 68 AGCTTAATTGTGAACATTTT 77 3153 3172 967 544060 54 73 GAAGGAGCTTAATTGTGAAC 59 3158 3177 968 544061 63 82 CAATAAAAAGAAGGAGCTTA 64 3167 3186 969 544062 66 85 GAACAATAAAAAGAAGGAGC 67 3170 3189 970 544063 85 104 CTGGAGGAAATAACTAGAGG 49 3189 3208 971 337460 88 107 ATTCTGGAGGAAATAACTAG 65 3192 3211 972 544064 112 131 TCAAATGATGAATTGTCTTG 58 3216 3235 973 544065 138 157 TTGATTTTGGCTCTGGAGAT 67 3242 3261 974 544066 145 164 GCAAATCTTGATTTTGGCTC 82 3249 3268 975 233676 148 167 ATAGCAAATCTTGATTTTGG 81 3252 3271 976 544067 156 175 CGTCTAACATAGCAAATCTT 87 3260 3279 977 544068 174 193 TGGCTAAAATTTTTACATCG 66 3278 3297 978 544069 178 197 CCATTGGCTAAAATTTTTAC 41 3282 3301 979 544070 184 203 AGGAGGCCATTGGCTAAAAT 36 3288 3307 980 544071 187 206 TGAAGGAGGCCATTGGCTAA 44 3291 3310 981 544072 195 214 GTCCCAACTGAAGGAGGCCA 59 3299 3318 982 544073 199 218 CCATGTCCCAACTGAAGGAG 54 3303 3322 983 544074 202 221 AGACCATGTCCCAACTGAAG 68 3306 3325 984 544075 206 225 TTTAAGACCATGTCCCAACT 51 3310 3329 985 544076 209 228 GTCTTTAAGACCATGTCCCA 64 3313 3332 986 544077 216 235 GGACAAAGTCTTTAAGACCA 45 3320 3339 987 544078 222 241 TCTTATGGACAAAGTCTTTA 40 3326 3345 988 544079 245 264 TATGTCATTAATTTGGCCCT 30 3349 3368 989 544080 270 289 GATCAAATATGTTGAGTTTT 65 3374 3393 990 233690 274 293 GACTGATCAAATATGTTGAG 75 3378 3397 991 544081 316 335 TCTTCTTTGATTTCACTGGT 86 3420 3439 992 544082 334 353 CTTCTCAGTTCCTTTTCTTC 69 3438 3457 993 544083 337 356 GTTCTTCTCAGTTCCTTTTC 77 3441 3460 994 544084 341 360 TGTAGTTCTTCTCAGTTCCT 75 3445 3464 995 544431 345 364 TATATGTAGTTCTTCTCAGT 15 3449 3468 996 544086 348 367 GTTTATATGTAGTTCTTCTC 65 3452 3471 997 544087 352 371 TGTAGTTTATATGTAGTTCT 49 3456 3475 998 544088 356 375 GACTTGTAGTTTATATGTAG 21 3460 3479 999 544089 364 383 TCATTTTTGACTTGTAGTTT 60 3468 3487 1000 544090 369 388 CCTCTTCATTTTTGACTTGT 83 3473 3492 1001 544091 375 394 TCTTTACCTCTTCATTTTTG 75 3479 3498 1002 544092 380 399 CATATTCTTTACCTCTTCAT 77 3484 3503 1003 544093 384 403 GTGACATATTCTTTACCTCT 76 3488 3507 1004 544094 392 411 GAGTTCAAGTGACATATTCT 71 3496 3515 1005 544095 398 417 TGAGTTGAGTTCAAGTGACA 44 3502 3521 1006 544096 403 422 AGTTTTGAGTTGAGTTCAAG 33 3507 3526 1007 544097 406 425 TCAAGTTTTGAGTTGAGTTC 69 3510 3529 1008 544098 414 433 GGAGGCTTTCAAGTTTTGAG 68 3518 3537 1009 544099 423 442 TTTCTTCTAGGAGGCTTTCA 79 3527 3546 1010 544100 427 446 ATTTTTTCTTCTAGGAGGCT 63 3531 3550 1011 544101 432 451 GTAGAATTTTTTCTTCTAGG 56 3536 3555 1012 544102 462 481 GCTCTTCTAAATATTTCACT 85 3566 3585 1013 544103 474 493 AGTTAGTTAGTTGCTCTTCT 71 3578 3597 1014 544104 492 511 CAGGTTGATTTTGAATTAAG 69 3596 3615 1015 544105 495 514 TTTCAGGTTGATTTTGAATT 53 3599 3618 1016 544106 499 518 GGAGTTTCAGGTTGATTTTG 64 3603 3622 1017 544107 504 523 GTTCTGGAGTTTCAGGTTGA 74 3608 3627 1018 544108 526 545 TTAAGTGAAGTTACTTCTGG 60 3630 3649 1019 544109 555 574 TGCTATTATCTTGTTTTTCT 63 4293 4312 1020 544110 564 583 GGTCTTTGATGCTATTATCT 65 4302 4321 1021 544111 567 586 GAAGGTCTTTGATGCTATTA 49 4305 4324 1022 544112 572 591 CTGGAGAAGGTCTTTGATGC 65 4310 4329 1023 544113 643 662 CTGAGCTGATTTTCTATTTC 64 N/A N/A 1024 337477 664 683 GGTTCTTGAATACTAGTCCT 82 6677 6696 234 544114 673 692 ATTTCTGTGGGTTCTTGAAT 57 6686 6705 1025 337478 675 694 AAATTTCTGTGGGTTCTTGA 29 6688 6707 235 544115 678 697 GAGAAATTTCTGTGGGTTCT 68 6691 6710 1026 544116 682 701 GATAGAGAAATTTCTGTGGG 54 6695 6714 1027 544117 689 708 CTTGGAAGATAGAGAAATTT 36 6702 6721 1028 337479 692 711 TGGCTTGGAAGATAGAGAAA 54 6705 6724 236 544118 699 718 GTGCTCTTGGCTTGGAAGAT 64 6712 6731 1029 544119 703 722 CTTGGTGCTCTTGGCTTGGA 68 6716 6735 1030 544120 707 726 AGTTCTTGGTGCTCTTGGCT 91 6720 6739 15 233710 710 729 AGTAGTTCTTGGTGCTCTTG 80 6723 6742 233 544121 713 732 GGGAGTAGTTCTTGGTGCTC 76 6726 6745 1031 544122 722 741 CTGAAGAAAGGGAGTAGTTC 55 6735 6754 1032 544123 752 771 ATCATGTTTTACATTTCTTA 52 6765 6784 1033 544124 755 774 GCCATCATGTTTTACATTTC 61 N/A N/A 1034 544125 759 778 GAATGCCATCATGTTTTACA 30 N/A N/A 1035 544126 762 781 CAGGAATGCCATCATGTTTT 34 N/A N/A 1036 337487 804 823 CACTTGTATGTTCACCTCTG 83 7389 7408 28 233717 889 908 TGAATTAATGTCCATGGACT 75 7876 7895 14 544202 2081 2100 AAAGTCAATGTGACTTAGTA 70 11053 11072 1037 544203 2104 2123 AAGGTATAGTGATACCTCAT 84 11076 11095 1038

TABLE 132 Inhibition of ANGPTL3 mRNA by 5-10-5 MOE gapmers targeting SEQ ID NO: 1 and 2 SEQ SEQ ID SEQ SEQ ID NO: NO: ID NO: ID NO: ISIS 1 Start 1 Stop % 2 Start 2 Stop SEQ NO Site Site Sequence inhibition Site Site ID NO 560535 N/A N/A ACTGTTTTCTTCTGGAAGCA 0 3102 3121 1039 560536 N/A N/A AAATAAGGTATAGTGATACC 0 11080 11099 1040 560537 N/A N/A ACAAATAAGGTATAGTGATA 1 11082 11101 1041 560538 N/A N/A TAACAAATAAGGTATAGTGA 0 11084 11103 1042 560539 N/A N/A TTTAACAAATAAGGTATAGT 16 11086 11105 1043 560540 N/A N/A ATATATTTTAACAAATAAGG 0 11092 11111 1044 560541 N/A N/A CAGTATATATTTTAACAAAT 0 11096 11115 1045 560542 N/A N/A TACAGTATATATTTTAACAA 0 11098 11117 1046 560543 N/A N/A TATACAGTATATATTTTAAC 0 11100 11119 1047 560544 N/A N/A ATAGTATTAAGTGTTAAAAT 0 11130 11149 1048 560545 N/A N/A TCATAGTATTAAGTGTTAAA 0 11132 11151 1049 560546 N/A N/A GTTTTCATAGTATTAAGTGT 26 11136 11155 1050 560547 N/A N/A ATTATTTGTTTTCATAGTAT 0 11143 11162 1051 560548 N/A N/A CTTTACAATTATTTGTTTTC 0 11150 11169 1052 560549 N/A N/A ATTCCTTTACAATTATTTGT 21 11154 11173 1053 560550 N/A N/A AGATTCCTTTACAATTATTT 18 11156 11175 1054 560551 N/A N/A CAAGATTCCTTTACAATTAT 21 11158 11177 1055 560552 N/A N/A GACAAGATTCCTTTACAATT 55 11160 11179 1056 560553 N/A N/A CTGACAAGATTCCTTTACAA 47 11162 11181 1057 560554 N/A N/A AATCTGACAAGATTCCTTTA 52 11165 11184 1058 560555 N/A N/A GTAATCTGACAAGATTCCTT 56 11167 11186 1059 560556 N/A N/A CTGTAATCTGACAAGATTCC 51 11169 11188 1060 560557 N/A N/A TACTGTAATCTGACAAGATT 18 11171 11190 1061 560558 N/A N/A CTTACTGTAATCTGACAAGA 33 11173 11192 1062 560559 N/A N/A TTCTTACTGTAATCTGACAA 47 11175 11194 1063 560560 N/A N/A CATTCTTACTGTAATCTGAC 65 11177 11196 1064 560561 N/A N/A TTCATTCTTACTGTAATCTG 54 11179 11198 1065 560562 N/A N/A TGTTCATTCTTACTGTAATC 44 11181 11200 1066 560563 N/A N/A TATGTTCATTCTTACTGTAA 39 11183 11202 1067 560564 N/A N/A AATATGTTCATTCTTACTGT 0 11185 11204 1068 560565 N/A N/A ACAAATATGTTCATTCTTAC 3 11188 11207 1069 560566 N/A N/A CCACAAATATGTTCATTCTT 75 11190 11209 42 560567 N/A N/A TGCCACAAATATGTTCATTC 80 11192 11211 43 560568 N/A N/A CGATGCCACAAATATGTTCA 64 11195 11214 1070 560569 N/A N/A CTCGATGCCACAAATATGTT 65 11197 11216 1071 560570 N/A N/A AACTCGATGCCACAAATATG 46 11199 11218 1072 560571 N/A N/A TTAACTCGATGCCACAAATA 52 11201 11220 1073 560572 N/A N/A CTTTAACTCGATGCCACAAA 66 11203 11222 1074 560573 N/A N/A AACTTTAACTCGATGCCACA 53 11205 11224 1075 560574 N/A N/A TAAACTTTAACTCGATGCCA 72 11207 11226 44 560575 N/A N/A AATATAAACTTTAACTCGAT 6 11211 11230 1076 560576 N/A N/A GAAATATAAACTTTAACTCG 17 11213 11232 1077 560577 N/A N/A GGGAAATATAAACTTTAACT 0 11215 11234 1078 560578 N/A N/A GAATCACAGCATATTTAGGG 46 11233 11252 1079 560579 N/A N/A TAGAATCACAGCATATTTAG 32 11235 11254 1080 560580 N/A N/A GTATTAGAATCACAGCATAT 51 11239 11258 1081 560581 N/A N/A ATGTATTAGAATCACAGCAT 64 11241 11260 1082 560582 N/A N/A GAATGTATTAGAATCACAGC 44 11243 11262 1083 560583 N/A N/A ACGAATGTATTAGAATCACA 44 11245 11264 1084 560584 N/A N/A ACACGAATGTATTAGAATCA 41 11247 11266 1085 560585 N/A N/A CTACACGAATGTATTAGAAT 15 11249 11268 1086 560586 N/A N/A ACCTACACGAATGTATTAGA 37 11251 11270 1087 560587 N/A N/A AAACCTACACGAATGTATTA 3 11253 11272 1088 560588 N/A N/A GAAAACCTACACGAATGTAT 27 11255 11274 1089 560589 N/A N/A TTGAAAACCTACACGAATGT 19 11257 11276 1090 560590 N/A N/A ACTTGAAAACCTACACGAAT 21 11259 11278 1091 560591 N/A N/A CTACTTGAAAACCTACACGA 43 11261 11280 1092 560592 N/A N/A TATTTCTACTTGAAAACCTA 29 11266 11285 1093 560593 N/A N/A TTTATTTCTACTTGAAAACC 2 11268 11287 1094 560594 N/A N/A GGTTTATTTCTACTTGAAAA 27 11270 11289 1095 560595 N/A N/A GAGGTTTATTTCTACTTGAA 45 11272 11291 1096 560596 N/A N/A ACGAGGTTTATTTCTACTTG 75 11274 11293 45 560597 N/A N/A TTACGAGGTTTATTTCTACT 49 11276 11295 1097 560598 N/A N/A TGTTACGAGGTTTATTTCTA 39 11278 11297 1098 560599 N/A N/A CTTGTTACGAGGTTTATTTC 32 11280 11299 1099 560600 N/A N/A AACTTGTTACGAGGTTTATT 27 11282 11301 1100 560601 N/A N/A GTAACTTGTTACGAGGTTTA 55 11284 11303 1101 560602 N/A N/A CAGTAACTTGTTACGAGGTT 51 11286 11305 1102 560603 N/A N/A TTCAGTAACTTGTTACGAGG 40 11288 11307 1103 560604 N/A N/A CGTTCAGTAACTTGTTACGA 53 11290 11309 1104 560605 N/A N/A CTTGTCAGGCTGTTTAAACG 24 11308 11327 1105 560606 N/A N/A TGCTTGTCAGGCTGTTTAAA 46 11310 11329 1106 560607 N/A N/A CATGCTTGTCAGGCTGTTTA 72 11312 11331 46 560608 N/A N/A TACATGCTTGTCAGGCTGTT 72 11314 11333 47 560609 N/A N/A TATACATGCTTGTCAGGCTG 63 11316 11335 1107 560610 N/A N/A TATATACATGCTTGTCAGGC 55 11318 11337 1108 560611 N/A N/A CATATATACATGCTTGTCAG 47 11320 11339 1109 560235 2 21 TGGAACTGTTTTCTTCTGGA 43 3106 3125 1110 337526 4 23 CGTGGAACTGTTTTCTTCTG 54 3108 3127 1111 560236 25 44 TTGATTTTCAATTTCAAGCA 91 3129 3148 30 560237 27 46 TCTTGATTTTCAATTTCAAG 33 3131 3150 1112 560238 32 51 TTTTATCTTGATTTTCAATT 0 3136 3155 1113 560239 35 54 CATTTTTATCTTGATTTTCA 6 3139 3158 1114 560240 43 62 ATTGTGAACATTTTTATCTT 0 3147 3166 1115 560241 45 64 TAATTGTGAACATTTTTATC 20 3149 3168 1116 560242 56 75 AAGAAGGAGCTTAATTGTGA 39 3160 3179 1117 560243 58 77 AAAAGAAGGAGCTTAATTGT 17 3162 3181 1118 560244 60 79 TAAAAAGAAGGAGCTTAATT 0 3164 3183 1119 560245 75 94 TAACTAGAGGAACAATAAAA 37 3179 3198 1120 560246 77 96 AATAACTAGAGGAACAATAA 3 3181 3200 1121 560247 79 98 GAAATAACTAGAGGAACAAT 13 3183 3202 1122 560248 81 100 AGGAAATAACTAGAGGAACA 28 3185 3204 1123 560249 83 102 GGAGGAAATAACTAGAGGAA 12 3187 3206 1124 560250 90 109 CAATTCTGGAGGAAATAACT 34 3194 3213 1125 560251 92 111 ATCAATTCTGGAGGAAATAA 32 3196 3215 1126 560252 96 115 CTTGATCAATTCTGGAGGAA 15 3200 3219 1127 560253 98 117 GTCTTGATCAATTCTGGAGG 53 3202 3221 1128 560254 100 119 TTGTCTTGATCAATTCTGGA 48 3204 3223 1129 560255 102 121 AATTGTCTTGATCAATTCTG 23 3206 3225 1130 560256 104 123 TGAATTGTCTTGATCAATTC 14 3208 3227 1131 560257 106 125 GATGAATTGTCTTGATCAAT 46 3210 3229 1132 560258 108 127 ATGATGAATTGTCTTGATCA 33 3212 3231 1133 560259 110 129 AAATGATGAATTGTCTTGAT 24 3214 3233 1134 560260 114 133 AATCAAATGATGAATTGTCT 25 3218 3237 1135 560261 116 135 AGAATCAAATGATGAATTGT 16 3220 3239 1136 560262 119 138 TAGAGAATCAAATGATGAAT 7 3223 3242 1137 560263 126 145 CTGGAGATAGAGAATCAAAT 40 3230 3249 1138 560264 128 147 CTCTGGAGATAGAGAATCAA 51 3232 3251 1139 560265 130 149 GGCTCTGGAGATAGAGAATC 63 3234 3253 31 560266 132 151 TTGGCTCTGGAGATAGAGAA 49 3236 3255 1140 560267 135 154 ATTTTGGCTCTGGAGATAGA 49 3239 3258 1141 560268 140 159 TCTTGATTTTGGCTCTGGAG 69 3244 3263 32 560269 142 161 AATCTTGATTTTGGCTCTGG 53 3246 3265 1142 560270 150 169 ACATAGCAAATCTTGATTTT 25 3254 3273 1143 560271 152 171 TAACATAGCAAATCTTGATT 0 3256 3275 1144 560272 154 173 TCTAACATAGCAAATCTTGA 53 3258 3277 1145 560273 176 195 ATTGGCTAAAATTTTTACAT 12 3280 3299 1146 560274 180 199 GGCCATTGGCTAAAATTTTT 34 3284 3303 1147 560275 182 201 GAGGCCATTGGCTAAAATTT 26 3286 3305 1148 560276 189 208 ACTGAAGGAGGCCATTGGCT 51 3293 3312 1149 560277 191 210 CAACTGAAGGAGGCCATTGG 28 3295 3314 1150 560278 193 212 CCCAACTGAAGGAGGCCATT 10 3297 3316 1151 560279 197 216 ATGTCCCAACTGAAGGAGGC 0 3301 3320 1152 560280 204 223 TAAGACCATGTCCCAACTGA 13 3308 3327 1153 560281 211 230 AAGTCTTTAAGACCATGTCC 4 3315 3334 1154 560282 213 232 CAAAGTCTTTAAGACCATGT 24 3317 3336 1155 560283 219 238 TATGGACAAAGTCTTTAAGA 8 3323 3342 1156 560284 224 243 CGTCTTATGGACAAAGTCTT 11 3328 3347 1157 560285 242 261 GTCATTAATTTGGCCCTTCG 57 3346 3365 33 560286 247 266 AATATGTCATTAATTTGGCC 0 3351 3370 1158 560287 249 268 GAAATATGTCATTAATTTGG 0 3353 3372 1159 560288 252 271 TTTGAAATATGTCATTAATT 4 3356 3375 1160 560289 256 275 AGTTTTTGAAATATGTCATT 7 3360 3379 1161 560290 258 277 TGAGTTTTTGAAATATGTCA 41 3362 3381 1162 560291 267 286 CAAATATGTTGAGTTTTTGA 30 3371 3390 1163 560292 272 291 CTGATCAAATATGTTGAGTT 32 3376 3395 1164 560293 276 295 AAGACTGATCAAATATGTTG 37 3380 3399 1165 560294 280 299 TAAAAAGACTGATCAAATAT 0 3384 3403 1166 560295 282 301 CATAAAAAGACTGATCAAAT 6 3386 3405 1167 560296 284 303 ATCATAAAAAGACTGATCAA 10 3388 3407 1168 560297 287 306 TAGATCATAAAAAGACTGAT 0 3391 3410 1169 560298 289 308 GATAGATCATAAAAAGACTG 21 3393 3412 1170 560299 291 310 GCGATAGATCATAAAAAGAC 20 3395 3414 1171 560300 293 312 CAGCGATAGATCATAAAAAG 16 3397 3416 1172 560301 295 314 TGCAGCGATAGATCATAAAA 38 3399 3418 1173 560302 297 316 TTTGCAGCGATAGATCATAA 32 3401 3420 1174 560303 299 318 GGTTTGCAGCGATAGATCAT 34 3403 3422 1175 560304 301 320 CTGGTTTGCAGCGATAGATC 25 3405 3424 1176 560305 303 322 CACTGGTTTGCAGCGATAGA 28 3407 3426 1177 560306 305 324 TTCACTGGTTTGCAGCGATA 65 3409 3428 34 560307 307 326 ATTTCACTGGTTTGCAGCGA 23 3411 3430 1178 560308 310 329 TTGATTTCACTGGTTTGCAG 5 3414 3433 1179 560309 318 337 CTTCTTCTTTGATTTCACTG 25 3422 3441 1180 560310 327 346 GTTCCTTTTCTTCTTCTTTG 19 3431 3450 1181 544120 707 726 AGTTCTTGGTGCTCTTGGCT 77 6720 6739 15 560311 801 820 TTGTATGTTCACCTCTGTTA 25 7386 7405 1182 560312 802 821 CTTGTATGTTCACCTCTGTT 37 7387 7406 1183 337487 804 823 CACTTGTATGTTCACCTCTG 83 7389 7408 28 560313 806 825 GCCACTTGTATGTTCACCTC 40 7391 7410 1184 560314 807 826 TGCCACTTGTATGTTCACCT 56 7392 7411 1185 560315 808 827 ATGCCACTTGTATGTTCACC 39 7393 7412 1186 337488 809 828 CATGCCACTTGTATGTTCAC 19 7394 7413 1187 560316 810 829 ACATGCCACTTGTATGTTCA 26 7395 7414 1188 560317 811 830 TACATGCCACTTGTATGTTC 20 7396 7415 1189 560318 814 833 GCATACATGCCACTTGTATG 2 7399 7418 1190 560319 815 834 GGCATACATGCCACTTGTAT 24 7400 7419 1191 560320 816 835 TGGCATACATGCCACTTGTA 7 7401 7420 1192 560321 817 836 ATGGCATACATGCCACTTGT 0 7402 7421 1193 560322 821 840 TCTGATGGCATACATGCCAC 26 7406 7425 1194 560323 822 841 GTCTGATGGCATACATGCCA 39 7407 7426 1195 560324 824 843 GGGTCTGATGGCATACATGC 15 7409 7428 1196 560325 825 844 TGGGTCTGATGGCATACATG 23 7410 7429 1197 560326 826 845 CTGGGTCTGATGGCATACAT 9 7411 7430 1198 560327 834 853 GAGAGTTGCTGGGTCTGATG 0 7419 7438 1199 560328 835 854 TGAGAGTTGCTGGGTCTGAT 2 7420 7439 1200 560329 836 855 TTGAGAGTTGCTGGGTCTGA 35 7421 7440 1201 560330 837 856 CTTGAGAGTTGCTGGGTCTG 17 7422 7441 1202 560331 838 857 ACTTGAGAGTTGCTGGGTCT 0 7423 7442 1203 560332 839 858 AACTTGAGAGTTGCTGGGTC 13 7424 7443 1204 560333 843 862 GAAAAACTTGAGAGTTGCTG 22 7428 7447 1205 560334 844 863 TGAAAAACTTGAGAGTTGCT 16 7429 7448 1206 560335 845 864 ATGAAAAACTTGAGAGTTGC 10 7430 7449 1207 560336 846 865 CATGAAAAACTTGAGAGTTG 2 7431 7450 1208 560337 851 870 GTAGACATGAAAAACTTGAG 13 7436 7455 1209 560338 853 872 CAGTAGACATGAAAAACTTG 3 7438 7457 1210 560339 861 880 TAACATCACAGTAGACATGA 30 7446 7465 1211 560340 862 881 ATAACATCACAGTAGACATG 34 7447 7466 1212 560341 863 882 TATAACATCACAGTAGACAT 0 7448 7467 1213 560342 864 883 ATATAACATCACAGTAGACA 10 7449 7468 1214 560343 865 884 GATATAACATCACAGTAGAC 9 7450 7469 1215 560344 866 885 TGATATAACATCACAGTAGA 20 7451 7470 1216 337490 867 886 CTGATATAACATCACAGTAG 24 7452 7471 1217 560345 868 887 CCTGATATAACATCACAGTA 36 7453 7472 1218 560346 869 888 ACCTGATATAACATCACAGT 35 7454 7473 1219 560347 870 889 TACCTGATATAACATCACAG 26 7455 7474 1220 560348 871 890 CTACCTGATATAACATCACA 38 N/A N/A 1221 560349 872 891 ACTACCTGATATAACATCAC 12 N/A N/A 1222 560350 873 892 GACTACCTGATATAACATCA 28 N/A N/A 1223 560351 874 893 GGACTACCTGATATAACATC 15 N/A N/A 1224 560352 875 894 TGGACTACCTGATATAACAT 0 N/A N/A 1225 560353 876 895 ATGGACTACCTGATATAACA 11 N/A N/A 1226 337491 877 896 CATGGACTACCTGATATAAC 3 N/A N/A 1227 560354 878 897 CCATGGACTACCTGATATAA 0 N/A N/A 1228 560355 879 898 TCCATGGACTACCTGATATA 13 N/A N/A 1229 560356 880 899 GTCCATGGACTACCTGATAT 50 N/A N/A 1230 560357 881 900 TGTCCATGGACTACCTGATA 12 N/A N/A 1231 560358 882 901 ATGTCCATGGACTACCTGAT 20 N/A N/A 1232 560359 883 902 AATGTCCATGGACTACCTGA 16 7870 7889 1233 560360 884 903 TAATGTCCATGGACTACCTG 26 7871 7890 1234 560361 885 904 TTAATGTCCATGGACTACCT 31 7872 7891 1235 560362 886 905 ATTAATGTCCATGGACTACC 42 7873 7892 1236 560363 887 906 AATTAATGTCCATGGACTAC 21 7874 7893 1237 560364 891 910 GTTGAATTAATGTCCATGGA 18 7878 7897 1238 560365 892 911 TGTTGAATTAATGTCCATGG 36 7879 7898 1239 560366 893 912 ATGTTGAATTAATGTCCATG 13 7880 7899 1240 560367 894 913 GATGTTGAATTAATGTCCAT 14 7881 7900 1241 560368 895 914 CGATGTTGAATTAATGTCCA 30 7882 7901 1242 560369 896 915 TCGATGTTGAATTAATGTCC 29 7883 7902 1243 560370 897 916 TTCGATGTTGAATTAATGTC 4 7884 7903 1244 560371 898 917 ATTCGATGTTGAATTAATGT 22 7885 7904 1245 560372 899 918 TATTCGATGTTGAATTAATG 0 7886 7905 1246 560373 900 919 CTATTCGATGTTGAATTAAT 0 7887 7906 1247 337492 901 920 TCTATTCGATGTTGAATTAA 59 7888 7907 29 560374 902 921 ATCTATTCGATGTTGAATTA 18 7889 7908 1248 560375 903 922 CATCTATTCGATGTTGAATT 27 7890 7909 1249 560376 904 923 CCATCTATTCGATGTTGAAT 40 7891 7910 1250 560377 905 924 TCCATCTATTCGATGTTGAA 23 7892 7911 1251 560378 906 925 ATCCATCTATTCGATGTTGA 47 7893 7912 1252 560379 907 926 GATCCATCTATTCGATGTTG 46 7894 7913 1253 560380 908 927 TGATCCATCTATTCGATGTT 16 7895 7914 1254 560381 909 928 GTGATCCATCTATTCGATGT 24 7896 7915 1255 560382 910 929 TGTGATCCATCTATTCGATG 21 7897 7916 1256 560383 911 930 TTGTGATCCATCTATTCGAT 19 7898 7917 1257 560384 1273 1292 TTTAGGTTGTTTTCTCCACA 35 10245 10264 1258 560385 1274 1293 ATTTAGGTTGTTTTCTCCAC 34 10246 10265 1259 560386 1278 1297 TACCATTTAGGTTGTTTTCT 15 10250 10269 1260 560387 1286 1305 GTTATATTTACCATTTAGGT 20 10258 10277 1261 560388 1287 1306 TGTTATATTTACCATTTAGG 17 10259 10278 1262 560389 1288 1307 TTGTTATATTTACCATTTAG 21 10260 10279 1263 560390 1289 1308 TTTGTTATATTTACCATTTA 4 10261 10280 1264 560391 1292 1311 TGGTTTGTTATATTTACCAT 23 10264 10283 1265 560392 1296 1315 CTCTTGGTTTGTTATATTTA 63 10268 10287 1266 560393 1297 1316 GCTCTTGGTTTGTTATATTT 61 10269 10288 1267 560394 1298 1317 TGCTCTTGGTTTGTTATATT 51 10270 10289 1268 560395 1301 1320 TTTTGCTCTTGGTTTGTTAT 2 10273 10292 1269 560396 1302 1321 ATTTTGCTCTTGGTTTGTTA 0 10274 10293 1270 560397 1303 1322 GATTTTGCTCTTGGTTTGTT 0 10275 10294 1271 560398 1304 1323 AGATTTTGCTCTTGGTTTGT 16 10276 10295 1272 560399 1305 1324 TAGATTTTGCTCTTGGTTTG 28 10277 10296 1273 560400 1307 1326 CTTAGATTTTGCTCTTGGTT 69 10279 10298 35 560401 1308 1327 GCTTAGATTTTGCTCTTGGT 77 10280 10299 36 560402 1309 1328 GGCTTAGATTTTGCTCTTGG 72 10281 10300 37 560403 1315 1334 CTCTCTGGCTTAGATTTTGC 38 10287 10306 1274 560404 1316 1335 CCTCTCTGGCTTAGATTTTG 49 10288 10307 1275 560405 1317 1336 TCCTCTCTGGCTTAGATTTT 46 10289 10308 1276 560406 1321 1340 CTTCTCCTCTCTGGCTTAGA 40 10293 10312 1277 560407 1322 1341 TCTTCTCCTCTCTGGCTTAG 57 10294 10313 1278 560408 1323 1342 CTCTTCTCCTCTCTGGCTTA 40 10295 10314 1279 337505 1328 1347 TAATCCTCTTCTCCTCTCTG 28 10300 10319 1280 560409 1329 1348 ATAATCCTCTTCTCCTCTCT 30 10301 10320 1281 560410 1330 1349 GATAATCCTCTTCTCCTCTC 9 10302 10321 1282 560411 1331 1350 AGATAATCCTCTTCTCCTCT 23 10303 10322 1283 560412 1332 1351 AAGATAATCCTCTTCTCCTC 12 10304 10323 1284 560413 1333 1352 CAAGATAATCCTCTTCTCCT 40 10305 10324 1285 560414 1334 1353 CCAAGATAATCCTCTTCTCC 52 10306 10325 1286 560415 1335 1354 TCCAAGATAATCCTCTTCTC 56 10307 10326 1287 560416 1336 1355 TTCCAAGATAATCCTCTTCT 60 10308 10327 1288 560417 1337 1356 CTTCCAAGATAATCCTCTTC 58 10309 10328 1289 560418 1338 1357 ACTTCCAAGATAATCCTCTT 31 10310 10329 1290 560419 1339 1358 GACTTCCAAGATAATCCTCT 52 10311 10330 1291 560420 1340 1359 AGACTTCCAAGATAATCCTC 49 10312 10331 1292 560421 1341 1360 GAGACTTCCAAGATAATCCT 56 10313 10332 1293 337506 1342 1361 TGAGACTTCCAAGATAATCC 49 10314 10333 1294 560422 1343 1362 TTGAGACTTCCAAGATAATC 34 10315 10334 1295 560423 1344 1363 TTTGAGACTTCCAAGATAAT 14 10316 10335 1296 560424 1345 1364 TTTTGAGACTTCCAAGATAA 27 10317 10336 1297 560425 1346 1365 ATTTTGAGACTTCCAAGATA 23 10318 10337 1298 560426 1348 1367 CCATTTTGAGACTTCCAAGA 40 10320 10339 1299 560427 1351 1370 CTTCCATTTTGAGACTTCCA 58 10323 10342 1300 560428 1355 1374 TAACCTTCCATTTTGAGACT 36 10327 10346 1301 560429 1356 1375 ATAACCTTCCATTTTGAGAC 51 10328 10347 1302 560430 1357 1376 TATAACCTTCCATTTTGAGA 33 10329 10348 1303 560431 1358 1377 GTATAACCTTCCATTTTGAG 53 10330 10349 1304 337508 1360 1379 GAGTATAACCTTCCATTTTG 28 10332 10351 1305 560432 1361 1380 AGAGTATAACCTTCCATTTT 50 10333 10352 1306 560433 1365 1384 TTATAGAGTATAACCTTCCA 63 10337 10356 1307 560434 1369 1388 GATTTTATAGAGTATAACCT 31 10341 10360 1308 560435 1370 1389 TGATTTTATAGAGTATAACC 6 10342 10361 1309 560436 1371 1390 TTGATTTTATAGAGTATAAC 14 10343 10362 1310 560437 1372 1391 GTTGATTTTATAGAGTATAA 2 10344 10363 1311 560438 1376 1395 TTTGGTTGATTTTATAGAGT 20 10348 10367 1312 560439 1386 1405 GGATCAACATTTTGGTTGAT 42 10358 10377 1313 560440 1387 1406 TGGATCAACATTTTGGTTGA 10 10359 10378 1314 560441 1388 1407 ATGGATCAACATTTTGGTTG 34 10360 10379 1315 560442 1398 1417 AATCTGTTGGATGGATCAAC 52 10370 10389 1316 560443 1399 1418 GAATCTGTTGGATGGATCAA 47 10371 10390 1317 560444 1403 1422 TTCTGAATCTGTTGGATGGA 30 10375 10394 1318 560445 1404 1423 TTTCTGAATCTGTTGGATGG 34 10376 10395 1319 560446 1405 1424 CTTTCTGAATCTGTTGGATG 50 10377 10396 1320 560447 1409 1428 AAAGCTTTCTGAATCTGTTG 29 10381 10400 1321 560448 1425 1444 TTGCCTCAGTTCATTCAAAG 38 10397 10416 1322 560449 1429 1448 AAATTTGCCTCAGTTCATTC 27 10401 10420 1323 560450 1434 1453 CTTTTAAATTTGCCTCAGTT 34 10406 10425 1324 560451 1440 1459 TATTGCCTTTTAAATTTGCC 21 10412 10431 1325 560452 1441 1460 TTATTGCCTTTTAAATTTGC 23 10413 10432 1326 560453 1446 1465 TTAAATTATTGCCTTTTAAA 1 10418 10437 1327 560454 1447 1466 TTTAAATTATTGCCTTTTAA 1 10419 10438 1328 560455 1448 1467 GTTTAAATTATTGCCTTTTA 48 10420 10439 1329 560456 1449 1468 TGTTTAAATTATTGCCTTTT 25 10421 10440 1330 560457 1450 1469 ATGTTTAAATTATTGCCTTT 0 10422 10441 1331 560458 1704 1723 TTTAATAAGTTCACCTATTG 26 10676 10695 1332 560459 1705 1724 ATTTAATAAGTTCACCTATT 26 10677 10696 1333 560460 1706 1725 TATTTAATAAGTTCACCTAT 16 10678 10697 1334 560461 1707 1726 TTATTTAATAAGTTCACCTA 4 10679 10698 1335 560462 1708 1727 GTTATTTAATAAGTTCACCT 36 10680 10699 1336 560463 1709 1728 AGTTATTTAATAAGTTCACC 0 10681 10700 1337 560464 1712 1731 AAAAGTTATTTAATAAGTTC 12 10684 10703 1338 560465 1719 1738 TATTTAGAAAAGTTATTTAA 0 10691 10710 1339 560466 1738 1757 TAAAAGTCTCTAAATTTTTT 0 10710 10729 1340 560467 1739 1758 ATAAAAGTCTCTAAATTTTT 0 10711 10730 1341 560468 1740 1759 AATAAAAGTCTCTAAATTTT 25 10712 10731 1342 560469 1760 1779 GCTCATATGATGCCTTTTAA 77 10732 10751 38 560470 1761 1780 AGCTCATATGATGCCTTTTA 73 10733 10752 39 560471 1762 1781 TAGCTCATATGATGCCTTTT 67 10734 10753 40 560472 1763 1782 TTAGCTCATATGATGCCTTT 42 10735 10754 1343 560473 1764 1783 ATTAGCTCATATGATGCCTT 61 10736 10755 1344 560474 1765 1784 TATTAGCTCATATGATGCCT 55 10737 10756 41 560475 1766 1785 ATATTAGCTCATATGATGCC 42 10738 10757 1345 560476 1767 1786 GATATTAGCTCATATGATGC 36 10739 10758 1346 560477 1768 1787 TGATATTAGCTCATATGATG 21 10740 10759 1347 560478 1769 1788 GTGATATTAGCTCATATGAT 40 10741 10760 1348 560479 1776 1795 GAAAGTTGTGATATTAGCTC 43 10748 10767 1349 560480 1777 1796 GGAAAGTTGTGATATTAGCT 19 10749 10768 1350 560481 1778 1797 GGGAAAGTTGTGATATTAGC 17 10750 10769 1351 560482 1779 1798 TGGGAAAGTTGTGATATTAG 29 10751 10770 1352 560483 1780 1799 CTGGGAAAGTTGTGATATTA 35 10752 10771 1353 560484 1781 1800 ACTGGGAAAGTTGTGATATT 25 10753 10772 1354 560485 1782 1801 AACTGGGAAAGTTGTGATAT 12 10754 10773 1355 560486 1783 1802 AAACTGGGAAAGTTGTGATA 21 10755 10774 1356 560487 1784 1803 TAAACTGGGAAAGTTGTGAT 22 10756 10775 1357 560488 1785 1804 TTAAACTGGGAAAGTTGTGA 12 10757 10776 1358 560489 1786 1805 TTTAAACTGGGAAAGTTGTG 22 10758 10777 1359 560490 1787 1806 TTTTAAACTGGGAAAGTTGT 23 10759 10778 1360 560491 1790 1809 GTTTTTTAAACTGGGAAAGT 1 10762 10781 1361 560492 1791 1810 AGTTTTTTAAACTGGGAAAG 0 10763 10782 1362 560493 1792 1811 TAGTTTTTTAAACTGGGAAA 0 10764 10783 1363 560494 1796 1815 GTACTAGTTTTTTAAACTGG 23 10768 10787 1364 560495 1799 1818 AGAGTACTAGTTTTTTAAAC 0 10771 10790 1365 560496 1801 1820 CAAGAGTACTAGTTTTTTAA 0 10773 10792 1366 560497 1806 1825 TTTAACAAGAGTACTAGTTT 21 10778 10797 1367 560498 1807 1826 TTTTAACAAGAGTACTAGTT 19 10779 10798 1368 560499 1808 1827 GTTTTAACAAGAGTACTAGT 37 10780 10799 1369 560500 1809 1828 AGTTTTAACAAGAGTACTAG 20 10781 10800 1370 560501 1810 1829 GAGTTTTAACAAGAGTACTA 21 10782 10801 1371 560502 1811 1830 AGAGTTTTAACAAGAGTACT 0 10783 10802 1372 560503 1814 1833 TTTAGAGTTTTAACAAGAGT 0 10786 10805 1373 560504 1815 1834 GTTTAGAGTTTTAACAAGAG 18 10787 10806 1374 560505 1817 1836 AAGTTTAGAGTTTTAACAAG 9 10789 10808 1375 560506 1818 1837 CAAGTTTAGAGTTTTAACAA 1 10790 10809 1376 560507 1822 1841 TAGTCAAGTTTAGAGTTTTA 21 10794 10813 1377 560508 1823 1842 TTAGTCAAGTTTAGAGTTTT 10 10795 10814 1378 560509 1824 1843 TTTAGTCAAGTTTAGAGTTT 20 10796 10815 1379 560510 1828 1847 TGTATTTAGTCAAGTTTAGA 8 10800 10819 1380 560511 1829 1848 CTGTATTTAGTCAAGTTTAG 37 10801 10820 1381 560512 1830 1849 TCTGTATTTAGTCAAGTTTA 46 10802 10821 1382 560513 1834 1853 GTCCTCTGTATTTAGTCAAG 38 10806 10825 1383 560514 1835 1854 AGTCCTCTGTATTTAGTCAA 29 10807 10826 1384 560515 1836 1855 CAGTCCTCTGTATTTAGTCA 47 10808 10827 1385 560516 1837 1856 CCAGTCCTCTGTATTTAGTC 31 10809 10828 1386 560517 1838 1857 ACCAGTCCTCTGTATTTAGT 31 10810 10829 1387 560518 1839 1858 TACCAGTCCTCTGTATTTAG 35 10811 10830 1388 560519 1840 1859 TTACCAGTCCTCTGTATTTA 30 10812 10831 1389 560520 1841 1860 ATTACCAGTCCTCTGTATTT 37 10813 10832 1390 560521 1842 1861 AATTACCAGTCCTCTGTATT 12 10814 10833 1391 560522 1843 1862 CAATTACCAGTCCTCTGTAT 38 10815 10834 1392 560523 1844 1863 ACAATTACCAGTCCTCTGTA 35 10816 10835 1393 560524 1845 1864 TACAATTACCAGTCCTCTGT 51 10817 10836 1394 560525 1846 1865 GTACAATTACCAGTCCTCTG 52 10818 10837 1395 560526 1847 1866 TGTACAATTACCAGTCCTCT 38 10819 10838 1396 560527 1848 1867 CTGTACAATTACCAGTCCTC 19 10820 10839 1397 560528 1849 1868 ACTGTACAATTACCAGTCCT 13 10821 10840 1398 560529 1850 1869 AACTGTACAATTACCAGTCC 27 10822 10841 1399 560530 1851 1870 GAACTGTACAATTACCAGTC 20 10823 10842 1400 560531 1852 1871 AGAACTGTACAATTACCAGT 24 10824 10843 1401 560532 1854 1873 TAAGAACTGTACAATTACCA 22 10826 10845 1402 560533 1855 1874 TTAAGAACTGTACAATTACC 20 10827 10846 1403 560534 1856 1875 TTTAAGAACTGTACAATTAC 1 10828 10847 1404

TABLE 133 Inhibition of ANGPTL3 mRNA by 5-10-5 MOE gapmers targeting SEQ ID NO: 1 and 2 SEQ SEQ SEQ ID SEQ ID ID NO: NO: ID NO: NO: 2 SEQ 1 Start 1 Stop % 2 Start Stop ID ISIS NO Site Site Sequence inhibition Site Site NO 544355 N/A N/A TTTCAGCATGTATCTCTTAA 69 7065 7084 21 544376 N/A N/A GGAGTGGTTCTTTTCACAGC 64 8298 8317 24 544380 N/A N/A TGGTCCTTTTAACTTCCAAT 50 8365 8384 25 560612 N/A N/A ACTTGAAATTATAATAGGAA 0 3798 3817 1405 560613 N/A N/A AAAAAACTAACTTGAAATTA 0 3807 3826 1406 560614 N/A N/A GAAACAAAAAACTAACTTGA 21 3812 3831 1407 560615 N/A N/A GTGTTTTCATATATAACATT 19 3835 3854 1408 560616 N/A N/A AATTTTCAGTGTTTTCATAT 0 3843 3862 1409 560617 N/A N/A AAAATGCAAATTTTCAGTGT 0 3851 3870 1410 560618 N/A N/A GTAATTTTCATATAAAATGC 0 3864 3883 1411 560619 N/A N/A GATTTGTAATTTTCATATAA 0 3869 3888 1412 560620 N/A N/A TAACCGATTTGTAATTTTCA 16 3874 3893 1413 560621 N/A N/A TAATTTAACCGATTTGTAAT 5 3879 3898 1414 560622 N/A N/A TTGTATAATTTAACCGATTT 13 3884 3903 1415 560623 N/A N/A CTAGATTGTATAATTTAACC 8 3889 3908 1416 560624 N/A N/A GTGTTCTAGATTGTATAATT 24 3894 3913 1417 560625 N/A N/A AATGACATAGTGTTCTAGAT 0 3903 3922 1418 560626 N/A N/A AGTGTAATGACATAGTGTTC 10 3908 3927 1419 560627 N/A N/A TTACAATAGTGTAATGACAT 0 3915 3934 1420 560628 N/A N/A TTCAGTAATTTACAATAGTG 12 3924 3943 1421 560629 N/A N/A TTACCTTCAGTAATTTACAA 9 3929 3948 1422 560630 N/A N/A TTAACTTTTTACTTACCTTC 7 3941 3960 1423 560631 N/A N/A GAATAGTTTTAAATTTTTTT 0 3960 3979 1424 560632 N/A N/A ACACTGGAGAATAGTTTTAA 10 3968 3987 1425 560633 N/A N/A TTTAAACACTGGAGAATAGT 0 3973 3992 1426 560634 N/A N/A TCTGTTTTAAACACTGGAGA 25 3978 3997 1427 560635 N/A N/A GTATTATTTAATCTGTTTTA 0 3989 4008 1428 560636 N/A N/A TTACTGTATTATTTAATCTG 5 3994 4013 1429 560637 N/A N/A TAAATCTTTTCCATTTACTG 18 4008 4027 1430 560638 N/A N/A ATGAATAAATCTTTTCCATT 12 4013 4032 1431 560639 N/A N/A GCATATTTTCATATGAATAA 9 4025 4044 1432 560640 N/A N/A GCCCAGCATATTTTCATATG 20 4030 4049 1433 560641 N/A N/A AAAAGAAAAAGCCCAGCATA 20 4040 4059 1434 560642 N/A N/A CTGAACTTCAATTAAAAGAA 5 4053 4072 1435 560643 N/A N/A GATTTTCTGAACTTCAATTA 9 4059 4078 1436 560644 N/A N/A TCTAAAATTTGATTTTCTGA 0 4069 4088 1437 560645 N/A N/A ACTATCTCTAAAATTTGATT 8 4075 4094 1438 560646 N/A N/A TTAAATTGTACTATCTCTAA 5 4084 4103 1439 560647 N/A N/A ACATTTTATTTAAATTGTAC 17 4093 4112 1440 560648 N/A N/A GTCCTTAACATTTTATTTAA 0 4100 4119 1441 560649 N/A N/A CATATTTTTGTCCTTAACAT 0 4109 4128 1442 560650 N/A N/A TAGCACATATTTTTGTCCTT 25 4114 4133 1443 560651 N/A N/A TCAAATAGCACATATTTTTG 0 4119 4138 1444 560652 N/A N/A CTTCTTTCAAATAGCACATA 41 4125 4144 1445 560653 N/A N/A CTTGTATGCTTCTTTCAAAT 19 4133 4152 1446 560654 N/A N/A ATTCCTTCCCCTTGTATGCT 12 4143 4162 1447 560655 N/A N/A TTGGCAATTCCTTCCCCTTG 36 4149 4168 1448 560656 N/A N/A GAATATTGGCAATTCCTTCC 38 4154 4173 1449 560657 N/A N/A TGAAAAATGAATATTGGCAA 0 4162 4181 1450 560658 N/A N/A TAATGGATTTGAAAAATGAA 0 4171 4190 1451 560659 N/A N/A ACTAATAATGGATTTGAAAA 1 4176 4195 1452 560660 N/A N/A CATAATCTAAATTTTTAAAC 6 4194 4213 1453 560661 N/A N/A CACTATCATAATCTAAATTT 4 4200 4219 1454 560662 N/A N/A AATTTCCTGTAACACTATCA 2 4212 4231 1455 560663 N/A N/A CTATTAATTTCCTGTAACAC 9 4217 4236 1456 560664 N/A N/A CTTTTCTATTAATTTCCTGT 5 4222 4241 1457 560665 N/A N/A CTCTTTCTTTTCTATTAATT 0 4228 4247 1458 560666 N/A N/A AGTTGCTTTCCTCTTTCTTT 0 4238 4257 1459 560667 N/A N/A TTATAAGTTGCTTTCCTCTT 10 4243 4262 1460 560668 N/A N/A GTTGGTTATAAGTTGCTTTC 6 4248 4267 1461 560669 N/A N/A AGTAGGTTGGTTATAAGTTG 4 4253 4272 1462 560670 N/A N/A TAGAGAGTAGGTTGGTTATA 0 4258 4277 1463 560671 N/A N/A GGATATAGAGAGTAGGTTGG 0 4263 4282 1464 560672 N/A N/A AGTCTGGATATAGAGAGTAG 0 4268 4287 1465 560673 N/A N/A TACAAAAGTCTGGATATAGA 7 4274 4293 1466 560674 N/A N/A GTTTTTCTACAAAAGTCTGG 12 4281 4300 1467 560675 N/A N/A TTACCTGATTTTCTATTTCT 15 4380 4399 1468 560676 N/A N/A ATACTGACTTACCTGATTTT 15 4388 4407 1469 560677 N/A N/A TTAAAATACTGACTTACCTG 2 4393 4412 1470 560678 N/A N/A TACCATTAAAATACTGACTT 0 4398 4417 1471 560679 N/A N/A GGACATACCATTAAAATACT 7 4403 4422 1472 560680 N/A N/A AAAGATGGGACATACCATTA 0 4410 4429 1473 560681 N/A N/A AGACCTGTGTGAAAGATGGG 19 4421 4440 1474 560682 N/A N/A TTTACAGACCTGTGTGAAAG 22 4426 4445 1475 560683 N/A N/A GTGTTTTTACAGACCTGTGT 47 4431 4450 1476 560684 N/A N/A ATTCAGTGTTTTTACAGACC 44 4436 4455 1477 560685 N/A N/A TTAGGATTCAGTGTTTTTAC 46 4441 4460 1478 560686 N/A N/A ATAATTTTAGGATTCAGTGT 15 4447 4466 1479 560687 N/A N/A GCTTGTAAATAATTTTAGGA 0 4455 4474 1480 560688 N/A N/A GTTAAAGCTTGTAAATAATT 0 4461 4480 1481 560689 N/A N/A TGTTTTATATCTCTTGAAAA 0 5571 5590 1482 560690 N/A N/A TTGGTAATAATATTTGTTTT 9 5585 5604 1483 560691 N/A N/A GGAAATTGGTAATAATATTT 0 5590 5609 1484 560692 N/A N/A TTAGTGGAAATTGGTAATAA 22 5595 5614 1485 560693 N/A N/A TTTGTTTAGTGGAAATTGGT 8 5600 5619 1486 560694 N/A N/A TTATGTTTGTTTAGTGGAAA 0 5605 5624 1487 560695 N/A N/A TAACATTATGTTTGTTTAGT 12 5610 5629 1488 560696 N/A N/A ACTACTAACATTATGTTTGT 4 5615 5634 1489 560697 N/A N/A GCAGCACTACTAACATTATG 38 5620 5639 1490 560698 N/A N/A TTTTAGCAGCACTACTAACA 15 5625 5644 1491 560699 N/A N/A AAACCTTTTAGCAGCACTAC 52 5630 5649 1492 560700 N/A N/A GATAAAAAACCTTTTAGCAG 0 5636 5655 1493 560701 N/A N/A TAGTTGATAAAAAACCTTTT 0 5641 5660 1494 560702 N/A N/A CAAAAGTAGTTGATAAAAAA 0 5647 5666 1495 560703 N/A N/A ATGGAAACCAAAAGTAGTTG 13 5655 5674 1496 560704 N/A N/A AAAGTATGGAAACCAAAAGT 20 5660 5679 1497 560705 N/A N/A GAAGGAAAGTATGGAAACCA 45 5665 5684 1498 560706 N/A N/A CATAAGAAGGAAAGTATGGA 10 5670 5689 1499 560707 N/A N/A TAACATCATAAGAAGGAAAG 0 5676 5695 1500 560708 N/A N/A GAATAATAACATCATAAGAA 0 5682 5701 1501 560709 N/A N/A GAATTTAGAATAATAACATC 1 5689 5708 1502 560710 N/A N/A TATAATTGAAAAGAATTTAG 8 5701 5720 1503 560711 N/A N/A TAGTAAAAGATATAATTGAA 0 5711 5730 1504 560712 N/A N/A AATCATAGTAAAAGATATAA 10 5716 5735 1505 560713 N/A N/A CAGGTTCATTTAATCATAGT 43 5727 5746 1506 560714 N/A N/A CTATAGTAACATTTTGCTTT 24 5753 5772 1507 560715 N/A N/A GTATATTACTATAGTAACAT 18 5761 5780 1508 560716 N/A N/A ACAATGTATATTACTATAGT 0 5766 5785 1509 560717 N/A N/A TAGACACAATGTATATTACT 46 5771 5790 1510 560718 N/A N/A TATTTTTAGACACAATGTAT 29 5777 5796 1511 560719 N/A N/A ACACATTTTTATTTTTAGAC 15 5786 5805 1512 560720 N/A N/A TTGGTTTCTTCACACATTTT 62 5797 5816 1513 560721 N/A N/A TTCATTGTTTTGGTTTCTTC 55 5806 5825 1514 560722 N/A N/A CAGAAATTCATTGTTTTGGT 55 5812 5831 1515 560723 N/A N/A TCCAACTCAGAAATTCATTG 65 5819 5838 48 560724 N/A N/A CTTCTTCCAACTCAGAAATT 41 5824 5843 1516 560725 N/A N/A TGATCTAACTCTTCTTCCAA 24 5834 5853 1517 560726 N/A N/A TTAAATGATCTAACTCTTCT 23 5839 5858 1518 560727 N/A N/A TGAGAAAGTTAAATGATCTA 0 5847 5866 1519 560728 N/A N/A TACTTAAATTTTTAGAGTTT 10 5886 5905 1520 560729 N/A N/A AAAGTTACTTAAATTTTTAG 3 5891 5910 1521 560730 N/A N/A ATCTTAAAGTTACTTAAATT 0 5896 5915 1522 560731 N/A N/A ATGTGATCTTAAAGTTACTT 24 5901 5920 1523 560732 N/A N/A TAACTATGTGATCTTAAAGT 0 5906 5925 1524 560733 N/A N/A TTACTCTTTTCTACTAAGTA 39 5924 5943 1525 560734 N/A N/A GGGTATTACTCTTTTCTACT 48 5929 5948 1526 560735 N/A N/A TTGCTGGGTATTACTCTTTT 75 5934 5953 49 560736 N/A N/A TTTGCTTGCTGGGTATTACT 65 5939 5958 50 560737 N/A N/A TAAAGTTTGCTTGCTGGGTA 49 5944 5963 1527 560738 N/A N/A TATTGTAAAGTTTGCTTGCT 15 5949 5968 1528 560739 N/A N/A TAAAAGGATCTATTGTAAAG 0 5959 5978 1529 560740 N/A N/A TTATTTAAAAGGATCTATTG 9 5964 5983 1530 560741 N/A N/A GGACCTTATTTAAAAGGATC 17 5969 5988 1531 560742 N/A N/A GATATTTCCTAGGACCTTAT 27 5980 5999 1532 560743 N/A N/A TGAATGATATTTCCTAGGAC 0 5985 6004 1533 560744 N/A N/A TGGCATGAATGATATTTCCT 74 5990 6009 51 560745 N/A N/A GATGCTGGCATGAATGATAT 40 5995 6014 1534 560746 N/A N/A TTTTTTGATGCTGGCATGAA 38 6001 6020 1535 560747 N/A N/A GTTAGTTTTTTGATGCTGGC 35 6006 6025 1536 560748 N/A N/A TTAGTGTTAGTTTTTTGATG 0 6011 6030 1537 560749 N/A N/A GCATTATTAGTGTTAGTTTT 50 6017 6036 1538 560750 N/A N/A ATCTTGCATTATTAGTGTTA 49 6022 6041 1539 560751 N/A N/A ATAATATCTTGCATTATTAG 17 6027 6046 1540 560752 N/A N/A CAGTAAGAAAAGCAGAATAT 15 6047 6066 1541 560753 N/A N/A TCATTGACAGTAAGAAAAGC 47 6054 6073 1542 560754 N/A N/A GATAGTTTTTCTCATTGACA 40 6065 6084 1543 560755 N/A N/A GTTTGCAATTTATTGAATGA 12 6083 6102 1544 560756 N/A N/A GTGTTGGGTTTGCAATTTAT 55 6090 6109 1545 560757 N/A N/A TTAAGTGTGTTGGGTTTGCA 50 6096 6115 1546 560758 N/A N/A TTTTATTTAAGTGTGTTGGG 5 6102 6121 1547 560759 N/A N/A TTTAGCAGTAACATTTTATT 19 6121 6140 1548 560760 N/A N/A GTTAGTTTAGCAGTAACATT 30 6126 6145 1549 560761 N/A N/A TCTATATATTCAGTAGTTTA 17 6148 6167 1550 560762 N/A N/A TTACTTTCTATATATTCAGT 14 6154 6173 1551 560763 N/A N/A GTTTGCTTACTTTCTATATA 20 6160 6179 1552 560764 N/A N/A AGTTTGTTTGCTTACTTTCT 36 6165 6184 1553 560765 N/A N/A TGGCAAGTTTGTTTGCTTAC 43 6170 6189 1554 560766 N/A N/A TTACTGTTACTGTATTTCCC 39 10155 10174 1555 560767 N/A N/A ATGTAGTTACTGTTACTGTA 18 10161 10180 1556 560768 N/A N/A ATTTAATGGGTACAGACTCG 47 10182 10201 61 560769 N/A N/A ATGCAATTTAATGGGTACAG 32 10187 10206 1557 560770 N/A N/A TAGATATGCAATTTAATGGG 4 10192 10211 1558 560771 N/A N/A AGGAGATAGATATGCAATTT 5 10198 10217 1559 560772 N/A N/A CCTAAAGGAGATAGATATGC 36 10203 10222 1560 560773 N/A N/A AGCCTCCTAAAGGAGATAGA 0 10208 10227 1561 560774 N/A N/A CACCACCAGCCTCCTAAAGG 35 10215 10234 1562 560775 N/A N/A ATCTAAGAAAATTAATAAAC 17 7003 7022 1563 560776 N/A N/A ATGATCACATCTAAGAAAAT 8 7011 7030 1564 560777 N/A N/A ATACCATGATCACATCTAAG 49 7016 7035 62 560778 N/A N/A GCAATACCATGATCACATCT 59 7019 7038 52 560779 N/A N/A AACTGCAATACCATGATCAC 35 7023 7042 1565 560780 N/A N/A TAAAACTGCAATACCATGAT 43 7026 7045 1566 560781 N/A N/A CTTTAAAACTGCAATACCAT 13 7029 7048 1567 560782 N/A N/A TCTCCTTTAAAACTGCAATA 18 7033 7052 1568 560783 N/A N/A TGTTCTCCTTTAAAACTGCA 13 7036 7055 1569 560784 N/A N/A GATTGTTCTCCTTTAAAACT 23 7039 7058 1570 560785 N/A N/A AGGAGATTGTTCTCCTTTAA 14 7043 7062 1571 560786 N/A N/A AACAGGAGATTGTTCTCCTT 0 7046 7065 1572 560787 N/A N/A TTAAACAGGAGATTGTTCTC 7 7049 7068 1573 560788 N/A N/A CTCTTAAACAGGAGATTGTT 10 7052 7071 1574 560789 N/A N/A ACTCCGTAAATATTTCAGCA 55 7077 7096 53 560790 N/A N/A CTTTAACTCCGTAAATATTT 22 7082 7101 1575 560791 N/A N/A GACCTTTAACTCCGTAAATA 54 7085 7104 63 560792 N/A N/A AGTGACCTTTAACTCCGTAA 35 7088 7107 1576 560793 N/A N/A GGAGTCCAGTGACCTTTAAC 15 7095 7114 1577 560794 N/A N/A TCTGGAGTCCAGTGACCTTT 46 7098 7117 64 560795 N/A N/A ACCAGTCTGGAGTCCAGTGA 8 7103 7122 1578 560796 N/A N/A TCATCTTACCAAACTATTTT 22 7169 7188 1579 560797 N/A N/A GAATCATCTTACCAAACTAT 39 7172 7191 1580 560798 N/A N/A TAAGAATCATCTTACCAAAC 35 7175 7194 1581 560799 N/A N/A ATGTAAGAATCATCTTACCA 52 7178 7197 65 560800 N/A N/A AAGAATGTAAGAATCATCTT 22 7182 7201 1582 560801 N/A N/A GTTATTTAAGAATGTAAGAA 0 7189 7208 1583 560802 N/A N/A CGTGTTATTTAAGAATGTAA 3 7192 7211 1584 560803 N/A N/A AGCATTTTTCTTAGATGGCG 48 7210 7229 66 560804 N/A N/A TAAAGCATTTTTCTTAGATG 0 7213 7232 1585 560805 N/A N/A TGTTAAAGCATTTTTCTTAG 0 7216 7235 1586 560806 N/A N/A TTTATGTTAAAGCATTTTTC 20 7220 7239 1587 560807 N/A N/A ATGTTTATGTTAAAGCATTT 8 7223 7242 1588 560808 N/A N/A GCATTTTTTCAGTAATGTTT 40 7237 7256 1589 560809 N/A N/A TGTAGCATTTTTTCAGTAAT 24 7241 7260 1590 560810 N/A N/A CAAATGTAGCATTTTTTCAG 0 7245 7264 1591 560811 N/A N/A TGGCAAATGTAGCATTTTTT 60 7248 7267 54 560812 N/A N/A AAGTTGTGGCAAATGTAGCA 26 7254 7273 1592 560813 N/A N/A ATGAAGTTGTGGCAAATGTA 11 7257 7276 1593 560814 N/A N/A TTTATGAAGTTGTGGCAAAT 36 7260 7279 1594 560815 N/A N/A CATTTTATGAAGTTGTGGCA 45 7263 7282 67 560816 N/A N/A TGACATTTTATGAAGTTGTG 16 7266 7285 1595 560817 N/A N/A CACTTGACATTTTATGAAGT 47 7270 7289 68 560818 N/A N/A CTTGAGATTTCACTTGACAT 18 7280 7299 1596 560819 N/A N/A TTTGGAGCTTGAGATTTCAC 0 7287 7306 1597 560820 N/A N/A ATCTTTGGAGCTTGAGATTT 0 7290 7309 1598 560821 N/A N/A AATATCTTTGGAGCTTGAGA 6 7293 7312 1599 560822 N/A N/A AATAATATCTTTGGAGCTTG 24 7296 7315 1600 560823 N/A N/A AGGAATAATATCTTTGGAGC 1 7299 7318 1601 560824 N/A N/A AATAGGAATAATATCTTTGG 0 7302 7321 1602 560825 N/A N/A AGTAATAGGAATAATATCTT 0 7305 7324 1603 560826 N/A N/A TTACATCAGATTTAGTAATA 0 7318 7337 1604 560827 N/A N/A AAATGTTATTACATCAGATT 0 7326 7345 1605 560828 N/A N/A ATAAAATGTTATTACATCAG 12 7329 7348 1606 560829 N/A N/A CCTAGAATCAATAAAATGTT 13 7339 7358 1607 560830 N/A N/A AGGAATGCCTAGAATCAATA 9 7346 7365 1608 560831 N/A N/A ATTCAGCAGGAATGCCTAGA 26 7353 7372 1609 560832 N/A N/A TACATTCAGCAGGAATGCCT 23 7356 7375 1610 560833 N/A N/A TTACCTGATATAACATCACA 30 7456 7475 1611 560834 N/A N/A GTTTTACCTGATATAACATC 6 7459 7478 1612 560835 N/A N/A CAGGTTTTACCTGATATAAC 4 7462 7481 1613 560836 N/A N/A TTAGACAGGTTTTACCTGAT 6 7467 7486 1614 560837 N/A N/A ATTCTCCTTAGACAGGTTTT 6 7474 7493 1615 560838 N/A N/A ACTGTCTATTCTCCTTAGAC 0 7481 7500 1616 560839 N/A N/A ACTACTGTCTATTCTCCTTA 17 7484 7503 1617 560840 N/A N/A ACTAACTACTGTCTATTCTC 0 7488 7507 1618 560841 N/A N/A TGAACTAACTACTGTCTATT 0 7491 7510 1619 560842 N/A N/A AGTTGAACTAACTACTGTCT 0 7494 7513 1620 560844 N/A N/A ATTAATTGATATGTAAAACG 0 8347 8366 1621 560845 N/A N/A CCAATTAATTGATATGTAAA 15 8350 8369 1622 560846 N/A N/A TCCTTTTAACTTCCAATTAA 29 8362 8381 1623 560847 N/A N/A TCCTGGTCCTTTTAACTTCC 58 8368 8387 69 560848 N/A N/A GTTTCCTGGTCCTTTTAACT 0 8371 8390 1624 560849 N/A N/A TCTGAGTTTCCTGGTCCTTT 36 8376 8395 1625 560850 N/A N/A ATGTCTGAGTTTCCTGGTCC 31 8379 8398 1626 560851 N/A N/A TGTATGTCTGAGTTTCCTGG 0 8382 8401 1627 560852 N/A N/A ATGTATACTGTATGTCTGAG 19 8390 8409 1628 560853 N/A N/A AAAATGTATACTGTATGTCT 12 8393 8412 1629 560854 N/A N/A TTTTAAAATGTATACTGTAT 0 8397 8416 1630 560855 N/A N/A CATACATTCTATATATTATA 29 8432 8451 1631 560856 N/A N/A AAGCCATACATTCTATATAT 38 8436 8455 55 560857 N/A N/A ATTATAAGCCATACATTCTA 6 8441 8460 1632 560858 N/A N/A TTCATTATAAGCCATACATT 0 8444 8463 1633 560859 N/A N/A TAATTCATTATAAGCCATAC 19 8447 8466 1634 560860 N/A N/A TGAGTTAACTAATTCATTAT 0 8456 8475 1635 560861 N/A N/A TTTGCATTGAGTTAACTAAT 26 8463 8482 1636 560862 N/A N/A TAATTTGCATTGAGTTAACT 0 8466 8485 1637 560863 N/A N/A GAATAATTTGCATTGAGTTA 0 8469 8488 1638 560864 N/A N/A ATAGAATAATTTGCATTGAG 0 8472 8491 1639 560865 N/A N/A AAAATAGAATAATTTGCATT 0 8475 8494 1640 560866 N/A N/A TTGTAATCAAAATAGAATAA 0 8483 8502 1641 560867 N/A N/A TATTTGTAATCAAAATAGAA 16 8486 8505 1642 560868 N/A N/A TACTATTTGTAATCAAAATA 0 8489 8508 1643 560869 N/A N/A TTTTACTATTTGTAATCAAA 0 8492 8511 1644 560870 N/A N/A GCTTATTTTACTATTTGTAA 0 8497 8516 1645 560871 N/A N/A CTTGCTTATTTTACTATTTG 0 8500 8519 1646 560872 N/A N/A TTATCTTGCTTATTTTACTA 1 8504 8523 1647 560873 N/A N/A GTTATTTTATCTTGCTTATT 0 8510 8529 1648 560874 N/A N/A AAACATCTGTTATTTTATCT 0 8518 8537 1649 560875 N/A N/A GGATTTTAAACATCTGTTAT 0 8525 8544 1650 560876 N/A N/A CTTTTTGGATTTTAAACATC 24 8531 8550 1651 560877 N/A N/A GTGCTTTTTGGATTTTAAAC 6 8534 8553 1652 560878 N/A N/A TTTTGTATGTGCTTTTTGGA 24 8542 8561 1653 560879 N/A N/A GACATCATTCATGGATTTTT 50 8558 8577 70 560880 N/A N/A AGTACTTAGACATCATTCAT 43 8566 8585 71 560881 N/A N/A TAAGTGAGTACTTAGACATC 17 8572 8591 1654 560882 N/A N/A TACTTTATAAGTGAGTACTT 0 8579 8598 1655 560883 N/A N/A TTCTACTTTATAAGTGAGTA 32 8582 8601 1656 560884 N/A N/A AATGTCTTCTACTTTATAAG 0 8588 8607 1657 560885 N/A N/A AATAATGAATGTCTTCTACT 9 8595 8614 1658 560886 N/A N/A TATAATAATGAATGTCTTCT 0 8598 8617 1659 560887 N/A N/A TGATATAATAATGAATGTCT 29 8601 8620 1660 560888 N/A N/A AAAATTTGATATAATAATGA 0 8607 8626 1661 560889 N/A N/A CATTTAAAAATTTGATATAA 0 8613 8632 1662 560890 N/A N/A GTACTGAGCATTTAAAAATT 8 8621 8640 1663 560891 N/A N/A GGTCAAATAGTACTGAGCAT 40 8630 8649 72 560892 N/A N/A AATGGTCAAATAGTACTGAG 23 8633 8652 1664 560893 N/A N/A TTAAATGGTCAAATAGTACT 17 8636 8655 1665 560894 N/A N/A AGTTTGAATACAAAATTTTT 0 8654 8673 1666 560895 N/A N/A GGTAGTTTGAATACAAAATT 38 8657 8676 73 560896 N/A N/A ACTGGTAGTTTGAATACAAA 0 8660 8679 1667 560897 N/A N/A TTCACTGGTAGTTTGAATAC 0 8663 8682 1668 560898 N/A N/A GCTTTCACTGGTAGTTTGAA 25 8666 8685 1669 560899 N/A N/A AGGGCTTTCACTGGTAGTTT 30 8669 8688 1670 560900 N/A N/A GGTAGGGCTTTCACTGGTAG 9 8672 8691 1671 560901 N/A N/A CTAGGTAGGGCTTTCACTGG 37 8675 8694 1672 560902 N/A N/A CTTCTAGGTAGGGCTTTCAC 32 8678 8697 1673 560903 N/A N/A TACCTTCTAGGTAGGGCTTT 26 8681 8700 1674 560904 N/A N/A GTATACCTTCTAGGTAGGGC 0 8684 8703 1675 560905 N/A N/A TGAGTATACCTTCTAGGTAG 15 8687 8706 1676 560906 N/A N/A CACTGAGTATACCTTCTAGG 36 8690 8709 1677 560907 N/A N/A TATCACTGAGTATACCTTCT 0 8693 8712 1678 560908 N/A N/A ACTTATCACTGAGTATACCT 28 8696 8715 1679 560909 N/A N/A ACAAAACTTATCACTGAGTA 32 8701 8720 1680 560910 N/A N/A GCTACAAAACTTATCACTGA 15 8704 8723 1681 560911 N/A N/A GGAGCTACAAAACTTATCAC 21 8707 8726 1682 560912 N/A N/A GATTTGGAGCTACAAAACTT 0 8712 8731 1683 560913 N/A N/A GAAGATTTGGAGCTACAAAA 0 8715 8734 1684 560914 N/A N/A CTATTAGAAGATTTGGAGCT 0 8721 8740 1685 560915 N/A N/A CACTCACTATTAGAAGATTT 33 8727 8746 1686 560916 N/A N/A TGTCAGCCTTTTATTTTGGG 0 8751 8770 1687 560917 N/A N/A ACCTGTCAGCCTTTTATTTT 11 8754 8773 1688 560918 N/A N/A TCGACTTACCTGTCAGCCTT 0 8761 8780 1689 560919 N/A N/A TTCTCGACTTACCTGTCAGC 0 8764 8783 1690 560920 N/A N/A GTATTCTCGACTTACCTGTC 0 8767 8786 1691 560921 N/A N/A TAACATCCATATACAGTCAA 25 9177 9196 1692 560922 N/A N/A TATTAACATCCATATACAGT 20 9180 9199 1693 560923 N/A N/A ATTTATTAACATCCATATAC 20 9183 9202 1694 560924 N/A N/A GCTATTTATTAACATCCATA 47 9186 9205 1695 560925 N/A N/A TCAGCTATTTATTAACATCC 58 9189 9208 56 560926 N/A N/A CTGTCAGCTATTTATTAACA 30 9192 9211 1696 560927 N/A N/A TTACTGTCAGCTATTTATTA 22 9195 9214 1697 560928 N/A N/A ACTTTACTGTCAGCTATTTA 27 9198 9217 1698 560929 N/A N/A TAAACTTTACTGTCAGCTAT 41 9201 9220 1699 560930 N/A N/A GGATAAACTTTACTGTCAGC 45 9204 9223 1700 560931 N/A N/A TATGGATAAACTTTACTGTC 15 9207 9226 1701 560932 N/A N/A TTATATGGATAAACTTTACT 0 9210 9229 1702 560933 N/A N/A TTGCAAGTCTTTATATGGAT 47 9220 9239 1703 560934 N/A N/A TATTTGCAAGTCTTTATATG 26 9223 9242 1704 560935 N/A N/A GAATATTTGCAAGTCTTTAT 4 9226 9245 1705 560936 N/A N/A GAGGAATATTTGCAAGTCTT 58 9229 9248 57 560937 N/A N/A GTAGAGGAATATTTGCAAGT 47 9232 9251 1706 560938 N/A N/A TTGGTAGAGGAATATTTGCA 65 9235 9254 58 560939 N/A N/A GTTACATTATTATAGATATT 33 9269 9288 1707 560940 N/A N/A TGTGTTACATTATTATAGAT 20 9272 9291 1708 560941 N/A N/A GAAATGTGTTACATTATTAT 0 9276 9295 1709 560942 N/A N/A ACCAGTGAAATGTGTTACAT 56 9282 9301 59 560943 N/A N/A TTCACCAGTGAAATGTGTTA 19 9285 9304 1710 560944 N/A N/A TGTTTCACCAGTGAAATGTG 41 9288 9307 1711 560945 N/A N/A ACATGTTTCACCAGTGAAAT 0 9291 9310 1712 560946 N/A N/A AAGACATGTTTCACCAGTGA 48 9294 9313 1713 560947 N/A N/A GACAAGACATGTTTCACCAG 28 9297 9316 1714 560948 N/A N/A TATGACAAGACATGTTTCAC 13 9300 9319 1715 560949 N/A N/A GCATATGACAAGACATGTTT 12 9303 9322 1716 560950 N/A N/A TAATGCATATGACAAGACAT 4 9307 9326 1717 560951 N/A N/A CTATAATGCATATGACAAGA 22 9310 9329 1718 560952 N/A N/A TTTCTATAATGCATATGACA 23 9313 9332 1719 560953 N/A N/A TCCTTTCTATAATGCATATG 16 9316 9335 1720 560954 N/A N/A TCTGATTATCCTTTCTATAA 32 9324 9343 1721 560955 N/A N/A AAGTCTGATTATCCTTTCTA 42 9327 9346 1722 560956 N/A N/A TGAAAGTCTGATTATCCTTT 51 9330 9349 60 560957 N/A N/A AACTGAAAGTCTGATTATCC 31 9333 9352 1723 560958 N/A N/A TATAACTGAAAGTCTGATTA 6 9336 9355 1724 560959 N/A N/A GTTAAAAATATTAATATAAC 3 9350 9369 1725 560960 N/A N/A TGTGCACAAAAATGTTAAAA 0 9363 9382 1726 560961 N/A N/A CTATGTGCACAAAAATGTTA 9 9366 9385 1727 560962 N/A N/A TAGCTATGTGCACAAAAATG 29 9369 9388 1728 560963 N/A N/A AGATAGCTATGTGCACAAAA 41 9372 9391 1729 560964 N/A N/A TGAAGATAGCTATGTGCACA 23 9375 9394 1730 560965 N/A N/A TATTGAAGATAGCTATGTGC 13 9378 9397 1731 560966 N/A N/A TTTTATTGAAGATAGCTATG 4 9381 9400 1732 560967 N/A N/A CAATTTTATTGAAGATAGCT 17 9384 9403 1733 560968 N/A N/A AAACAATTTTATTGAAGATA 27 9387 9406 1734 560969 N/A N/A GTGTATCTTAAAATAATACC 7 9412 9431 1735 560970 N/A N/A TTAGTGTATCTTAAAATAAT 25 9415 9434 1736 560971 N/A N/A TGATCATTTTAGTGTATCTT 34 9423 9442 1737 560972 N/A N/A CCCTTGATCATTTTAGTGTA 7 9427 9446 1738 560973 N/A N/A AATCCCTTGATCATTTTAGT 0 9430 9449 1739 560974 N/A N/A TTGAATCCCTTGATCATTTT 20 9433 9452 1740 560975 N/A N/A TTAGTCTTGAATCCCTTGAT 28 9439 9458 1741 560976 N/A N/A TTGTTTAGTCTTGAATCCCT 40 9443 9462 1742 560977 N/A N/A GAGTTGTTTAGTCTTGAATC 6 9446 9465 1743 560978 N/A N/A ATTGAGTTGTTTAGTCTTGA 14 9449 9468 1744 560979 N/A N/A CTAATTGAGTTGTTTAGTCT 0 9452 9471 1745 560980 N/A N/A CAACTAATTGAGTTGTTTAG 0 9455 9474 1746 560981 N/A N/A ATTGGTGCAACTAATTGAGT 0 9462 9481 1747 560982 N/A N/A TTTATTGGTGCAACTAATTG 9 9465 9484 1748 560983 N/A N/A TTTTTTATTGGTGCAACTAA 8 9468 9487 1749 560984 N/A N/A TAAGTGTTTTTTATTGGTGC 20 9474 9493 1750 560985 N/A N/A ACTGACAGTTTTTTTAAGTG 16 9488 9507 1751 560986 N/A N/A GACACTGACAGTTTTTTTAA 6 9491 9510 1752 560987 N/A N/A TTGGACACTGACAGTTTTTT 0 9494 9513 1753 560988 N/A N/A AGGTTGGACACTGACAGTTT 6 9497 9516 1754 560989 N/A N/A TACAGGTTGGACACTGACAG 0 9500 9519 1755 544120 707 726 AGTTCTTGGTGCTCTTGGCT 72 6720 6739 15 337487 804 823 CACTTGTATGTTCACCTCTG 80 7389 7408 28 544145 1055 1074 GTTGTCTTTCCAGTCTTCCA 69 9630 9649 16 544156 1195 1214 GCTTTGTGATCCCAAGTAGA 61 9770 9789 17 544162 1269 1288 GGTTGTTTTCTCCACACTCA 71 10241 10260 18 544166 1353 1372 ACCTTCCATTTTGAGACTTC 65 10325 10344 19 544199 1907 1926 TACACATACTCTGTGCTGAC 69 10879 10898 20

TABLE 134 Inhibition of ANGPTL3 mRNA by 5-10-5 MOE gapmers targeting SEQ ID NO: 1 and 2 SEQ SEQ SEQ ID SEQ ID ID NO: NO: ID NO: NO: 2 SEQ ISIS 1 Start 1 Stop % 2 Start Stop ID NO Site Site Sequence inhibition Site Site NO 563720 N/A N/A TATATTGGATAATTTGAAAT 7 11610 11629 1756 563721 N/A N/A ATGTATATTGGATAATTTGA 17 11613 11632 1757 563722 N/A N/A GACATGTATATTGGATAATT 20 11616 11635 1758 563723 N/A N/A ATGACATGTATATTGGATAA 29 11618 11637 1759 563724 N/A N/A TATATATGACATGTATATTG 9 11623 11642 1760 563725 N/A N/A ATGTGACATATAAAAATATA 4 11639 11658 1761 563726 N/A N/A ATATGTGACATATAAAAATA 0 11641 11660 1762 563727 N/A N/A TTTATATATGTGACATATAA 0 11646 11665 1763 563728 N/A N/A CTTTTATATATGTGACATAT 16 11648 11667 1764 563729 N/A N/A ATCTTTTATATATGTGACAT 13 11650 11669 1765 563730 N/A N/A CATATCTTTTATATATGTGA 2 11653 11672 1766 563731 N/A N/A TCATACATATCTTTTATATA 2 11658 11677 1767 563732 N/A N/A TAGATCATACATATCTTTTA 31 11662 11681 1768 563733 N/A N/A CATAGATCATACATATCTTT 28 11664 11683 1769 563734 N/A N/A CACATAGATCATACATATCT 56 11666 11685 1770 563735 N/A N/A AGGATTCACATAGATCATAC 56 11672 11691 1771 563736 N/A N/A TTAGGATTCACATAGATCAT 24 11674 11693 1772 563737 N/A N/A ACTTAGGATTCACATAGATC 49 11676 11695 1773 563738 N/A N/A TTACTTAGGATTCACATAGA 15 11678 11697 1774 563739 N/A N/A TATTTACTTAGGATTCACAT 6 11681 11700 1775 563740 N/A N/A AATATTTACTTAGGATTCAC 28 11683 11702 1776 563741 N/A N/A TGTACTTTTCTGGAACAAAA 63 11701 11720 1777 563742 N/A N/A GATTATTTTTACCTTTATTA 21 11724 11743 1778 563743 N/A N/A TAGATTATTTTTACCTTTAT 5 11726 11745 1779 563744 N/A N/A ATTATAGATTATTTTTACCT 12 11730 11749 1780 563745 N/A N/A GAAAATTATAGATTATTTTT 15 11734 11753 1781 563746 N/A N/A GGTCCTGAAAATTATAGATT 7 11740 11759 1782 563747 N/A N/A GTGGTCCTGAAAATTATAGA 29 11742 11761 1783 563748 N/A N/A CTGTGGTCCTGAAAATTATA 37 11744 11763 1784 563749 N/A N/A GTCTGTGGTCCTGAAAATTA 47 11746 11765 1785 563750 N/A N/A TCGACAGCTTAGTCTGTGGT 66 11757 11776 1786 563751 N/A N/A TTTCGACAGCTTAGTCTGTG 41 11759 11778 1787 563752 N/A N/A AATTTCGACAGCTTAGTCTG 40 11761 11780 1788 563753 N/A N/A TTAATTTCGACAGCTTAGTC 35 11763 11782 1789 563754 N/A N/A CGTTAATTTCGACAGCTTAG 50 11765 11784 1790 563755 N/A N/A TGGCCCTAAAAAAATCAGCG 7 11783 11802 1791 563756 N/A N/A TCTGGCCCTAAAAAAATCAG 0 11785 11804 1792 563757 N/A N/A TGGTATTCTGGCCCTAAAAA 37 11791 11810 1793 563758 N/A N/A TTTGGTATTCTGGCCCTAAA 29 11793 11812 1794 563759 N/A N/A CCATTTTGGTATTCTGGCCC 35 11797 11816 1795 563760 N/A N/A GAGGAGCCATTTTGGTATTC 34 11803 11822 1796 563761 N/A N/A GAGAGGAGCCATTTTGGTAT 18 11805 11824 1797 563762 N/A N/A AAGAGAGGAGCCATTTTGGT 17 11807 11826 1798 563763 N/A N/A TGAAATTGTCCAATTTTGGG 28 11829 11848 1799 563764 N/A N/A TTTGAAATTGTCCAATTTTG 10 11831 11850 1800 563765 N/A N/A CATTTGAAATTGTCCAATTT 22 11833 11852 1801 563766 N/A N/A TGCATTTGAAATTGTCCAAT 45 11835 11854 1802 563767 N/A N/A ATTTTGCATTTGAAATTGTC 35 11839 11858 1803 563768 N/A N/A ATAATGAATTATTTTGCATT 0 11849 11868 1804 563769 N/A N/A TAAATAATGAATTATTTTGC 17 11852 11871 1805 563770 N/A N/A CTCATATATTAAATAATGAA 0 11861 11880 1806 563771 N/A N/A AACTCATATATTAAATAATG 16 11863 11882 1807 563772 N/A N/A TAGAGGAAGCAACTCATATA 7 11873 11892 1808 563773 N/A N/A AATAGAGGAAGCAACTCATA 20 11875 11894 1809 563774 N/A N/A CAAATAGAGGAAGCAACTCA 29 11877 11896 1810 563775 N/A N/A ACCAAATAGAGGAAGCAACT 27 11879 11898 1811 563776 N/A N/A AAACCAAATAGAGGAAGCAA 22 11881 11900 1812 563777 N/A N/A GGAAACCAAATAGAGGAAGC 37 11883 11902 1813 563778 N/A N/A TAAGGAAACCAAATAGAGGA 0 11886 11905 1814 563779 N/A N/A TTTAAGGAAACCAAATAGAG 0 11888 11907 1815 563780 N/A N/A TGTTTTCTTCTGGAAGCAGA 5 3100 3119 1816 563781 N/A N/A CTTACTTTAAGTGAAGTTAC 0 3636 3655 1817 563782 N/A N/A TTTTCTACTTACTTTAAGTG 3 3643 3662 1818 563783 N/A N/A ACATGAACCCTCTTTATTTT 0 3659 3678 1819 563784 N/A N/A GAAAACATAAACATGAACCC 0 3669 3688 1820 563785 N/A N/A AGATCCACATTGAAAACATA 8 3680 3699 1821 563786 N/A N/A TTAAAAGATCCACATTGAAA 8 3685 3704 1822 563787 N/A N/A GCCTTAGAAATATTTTTTTT 2 3703 3722 1823 563788 N/A N/A CAAATGGCATGCCTTAGAAA 29 3713 3732 1824 563789 N/A N/A TATTTCAAATGGCATGCCTT 24 3718 3737 1825 563790 N/A N/A CAAAGTATTTCAAATGGCAT 8 3723 3742 1826 563791 N/A N/A TGCAACAAAGTATTTCAAAT 0 3728 3747 1827 563792 N/A N/A TCAACAATGCAACAAAGTAT 3 3735 3754 1828 563793 N/A N/A GAAAAAAAAGTATTTCAACA 4 3749 3768 1829 563794 N/A N/A GATTATTTTTCTTGGAAAAA 11 3763 3782 1830 563795 N/A N/A GAAATTTTATTTTCTGGAGA 10 3781 3800 1831 563796 N/A N/A AAATTATAATAGGAAATTTT 14 3793 3812 1832 563797 N/A N/A CTGAATATAATGAATGAAAT 1 7854 7873 1833 563798 N/A N/A TACCTGAATATAATGAATGA 4 7857 7876 1834 563799 N/A N/A GACTACCTGAATATAATGAA 25 7860 7879 1835 563800 N/A N/A ATGGACTACCTGAATATAAT 15 7863 7882 1836 563801 N/A N/A TCCATGGACTACCTGAATAT 39 7866 7885 1837 563802 N/A N/A ACCATCAAGCCTCCCAAAAC 23 7952 7971 1838 563803 N/A N/A CCTTACCATCAAGCCTCCCA 29 7956 7975 1839 563804 N/A N/A AGTCCCCTTACCATCAAGCC 31 7961 7980 1840 563805 N/A N/A TGTAGTCCCCTTACCATCAA 18 7964 7983 1841 563806 N/A N/A GAATGTAGTCCCCTTACCAT 0 7967 7986 1842 563807 N/A N/A ATTGAATGTAGTCCCCTTAC 12 7970 7989 1843 563808 N/A N/A ATGATTGAATGTAGTCCCCT 14 7973 7992 1844 563809 N/A N/A GATTAGCAAGTGAATGAATG 13 7990 8009 1845 563810 N/A N/A GTAGATTAGCAAGTGAATGA 25 7993 8012 1846 563811 N/A N/A TTTGTAGATTAGCAAGTGAA 9 7996 8015 1847 563812 N/A N/A ATATTTGTAGATTAGCAAGT 0 7999 8018 1848 563813 N/A N/A CCATAAGAGGTTCTCAGTAA 44 8019 8038 1849 563814 N/A N/A GGTCCATAAGAGGTTCTCAG 37 8022 8041 1850 563815 N/A N/A CCTGGTCCATAAGAGGTTCT 25 8025 8044 1851 563816 N/A N/A TAATACCTGGTCCATAAGAG 9 8030 8049 1852 563817 N/A N/A TCCTAATACCTGGTCCATAA 39 8033 8052 1853 563818 N/A N/A TTTTCCTAATACCTGGTCCA 43 8036 8055 1854 563819 N/A N/A TACTTTTCCTAATACCTGGT 43 8039 8058 1855 563820 N/A N/A CGTTACTACTTTTCCTAATA 47 8045 8064 1856 563821 N/A N/A AAGGCTGAGACTGCTTCTCG 46 8067 8086 1857 563822 N/A N/A GATAATAAATTATATGAAGG 5 8083 8102 1858 563823 N/A N/A GTTTGATAATAAATTATATG 0 8087 8106 1859 563824 N/A N/A GTGTAATTGTTTGATAATAA 14 8095 8114 1860 563825 N/A N/A AATGTGTAATTGTTTGATAA 0 8098 8117 1861 563826 N/A N/A GTAATTTACTAACAAATGTG 18 8112 8131 1862 563827 N/A N/A AGTGTAATTTACTAACAAAT 0 8115 8134 1863 563828 N/A N/A ATAAGTGTAATTTACTAACA 0 8118 8137 1864 563829 N/A N/A GTAATAAGTGTAATTTACTA 0 8121 8140 1865 563830 N/A N/A GTTGTAATAAGTGTAATTTA 20 8124 8143 1866 563831 N/A N/A ACAGTTGTAATAAGTGTAAT 1 8127 8146 1867 563832 N/A N/A ATAACAGTTGTAATAAGTGT 4 8130 8149 1868 563833 N/A N/A TTCAAATAATAACAGTTGTA 0 8138 8157 1869 563834 N/A N/A ATAATTCAAATAATAACAGT 16 8142 8161 1870 563835 N/A N/A AATTGTGATAAATATAATTC 0 8155 8174 1871 563836 N/A N/A ATGTAATTGTGATAAATATA 0 8159 8178 1872 563837 N/A N/A GACATGTAATTGTGATAAAT 8 8162 8181 1873 563838 N/A N/A ACAGACATGTAATTGTGATA 33 8165 8184 1874 563839 N/A N/A AGAACAGACATGTAATTGTG 34 8168 8187 1875 563840 N/A N/A TTAAGAACAGACATGTAATT 0 8171 8190 1876 563841 N/A N/A AAGTATATTTAAGAACAGAC 0 8179 8198 1877 563842 N/A N/A TTAAATTGTGATAAGTATAT 1 8191 8210 1878 563843 N/A N/A GAATTAAATTGTGATAAGTA 0 8194 8213 1879 563844 N/A N/A GTGGAATTAAATTGTGATAA 0 8197 8216 1880 563845 N/A N/A GCCGTGGAATTAAATTGTGA 20 8200 8219 1881 563846 N/A N/A TAAGCCGTGGAATTAAATTG 16 8203 8222 1882 563847 N/A N/A TTGTAAGCCGTGGAATTAAA 28 8206 8225 1883 563848 N/A N/A TCATTGTAAGCCGTGGAATT 25 8209 8228 1884 563849 N/A N/A TGATCATTGTAAGCCGTGGA 49 8212 8231 1885 563850 N/A N/A TATAGTTATGATCATTGTAA 0 8220 8239 1886 563851 N/A N/A AATTATAGTTATGATCATTG 0 8223 8242 1887 563852 N/A N/A CTTTAATAATTATAGTTATG 0 8230 8249 1888 563853 N/A N/A TGTCTTTAATAATTATAGTT 4 8233 8252 1889 563854 N/A N/A AATTGTCTTTAATAATTATA 0 8236 8255 1890 563855 N/A N/A TCAAAATTGTCTTTAATAAT 7 8240 8259 1891 563856 N/A N/A ATTTAATCAAAATTGTCTTT 0 8246 8265 1892 563857 N/A N/A TAACATTTAATCAAAATTGT 0 8250 8269 1893 563858 N/A N/A ACATAACATTTAATCAAAAT 0 8253 8272 1894 563859 N/A N/A ATGACATAACATTTAATCAA 13 8256 8275 1895 563860 N/A N/A TACTTATGACATAACATTTA 0 8261 8280 1896 563861 N/A N/A TTACTACTTATGACATAACA 0 8265 8284 1897 563862 N/A N/A AACAGTTACTACTTATGACA 31 8270 8289 1898 563863 N/A N/A TGTAACAGTTACTACTTATG 29 8273 8292 1899 563864 N/A N/A CTTATTTGTAACAGTTACTA 0 8279 8298 1900 563865 N/A N/A TTTCACAGCTTATTTGTAAC 29 8287 8306 1901 563866 N/A N/A TCTTTTCACAGCTTATTTGT 22 8290 8309 1902 563867 N/A N/A GGTTCTTTTCACAGCTTATT 66 8293 8312 1903 563868 N/A N/A CTAGGAGTGGTTCTTTTCAC 37 8301 8320 1904 563869 N/A N/A ATGCTAGGAGTGGTTCTTTT 20 8304 8323 1905 563870 N/A N/A CTAATGCTAGGAGTGGTTCT 30 8307 8326 1906 563871 N/A N/A AGAGTGACTAATGCTAGGAG 41 8314 8333 1907 563872 N/A N/A AGAGAATAGAGTGACTAATG 28 8321 8340 1908 563873 N/A N/A TTAATGAGAGAATAGAGTGA 4 8327 8346 1909 563496 608 627 CTGTTGGTTTAATTGTTTAT 33 4346 4365 1910 563497 610 629 TGCTGTTGGTTTAATTGTTT 29 4348 4367 1911 563498 612 631 TATGCTGTTGGTTTAATTGT 27 4350 4369 1912 563499 614 633 ACTATGCTGTTGGTTTAATT 24 4352 4371 1913 563500 616 635 TGACTATGCTGTTGGTTTAA 68 4354 4373 1914 563501 619 638 ATTTGACTATGCTGTTGGTT 45 4357 4376 1915 563502 621 640 TTATTTGACTATGCTGTTGG 39 4359 4378 1916 563503 623 642 TTTTATTTGACTATGCTGTT 33 4361 4380 1917 563504 625 644 TCTTTTATTTGACTATGCTG 55 4363 4382 1918 563505 627 646 TTTCTTTTATTTGACTATGC 29 4365 4384 1919 563506 646 665 CTTCTGAGCTGATTTTCTAT 40 N/A N/A 1920 563507 648 667 TCCTTCTGAGCTGATTTTCT 76 N/A N/A 1921 563508 650 669 AGTCCTTCTGAGCTGATTTT 37 N/A N/A 1922 563509 652 671 CTAGTCCTTCTGAGCTGATT 52 N/A N/A 1923 563510 654 673 TACTAGTCCTTCTGAGCTGA 52 6667 6686 1924 563511 656 675 AATACTAGTCCTTCTGAGCT 41 6669 6688 1925 563512 658 677 TGAATACTAGTCCTTCTGAG 55 6671 6690 1926 563513 660 679 CTTGAATACTAGTCCTTCTG 43 6673 6692 1927 563514 662 681 TTCTTGAATACTAGTCCTTC 34 6675 6694 1928 563515 666 685 TGGGTTCTTGAATACTAGTC 52 6679 6698 1929 563516 668 687 TGTGGGTTCTTGAATACTAG 34 6681 6700 1930 563517 670 689 TCTGTGGGTTCTTGAATACT 43 6683 6702 1931 563518 680 699 TAGAGAAATTTCTGTGGGTT 0 6693 6712 1932 563519 684 703 AAGATAGAGAAATTTCTGTG 4 6697 6716 1933 563520 686 705 GGAAGATAGAGAAATTTCTG 0 6699 6718 1934 563521 694 713 CTTGGCTTGGAAGATAGAGA 29 6707 6726 1935 563522 696 715 CTCTTGGCTTGGAAGATAGA 51 6709 6728 1936 563523 705 724 TTCTTGGTGCTCTTGGCTTG 63 6718 6737 75 544120 707 726 AGTTCTTGGTGCTCTTGGCT 86 6720 6739 15 563524 715 734 AAGGGAGTAGTTCTTGGTGC 44 6728 6747 1937 563525 716 735 AAAGGGAGTAGTTCTTGGTG 14 6729 6748 1938 563526 717 736 GAAAGGGAGTAGTTCTTGGT 33 6730 6749 1939 563527 718 737 AGAAAGGGAGTAGTTCTTGG 0 6731 6750 1940 563528 719 738 AAGAAAGGGAGTAGTTCTTG 0 6732 6751 1941 563529 720 739 GAAGAAAGGGAGTAGTTCTT 0 6733 6752 1942 563530 726 745 TCAACTGAAGAAAGGGAGTA 0 6739 6758 1943 337481 728 747 ATTCAACTGAAGAAAGGGAG 23 6741 6760 1944 563531 729 748 CATTCAACTGAAGAAAGGGA 16 6742 6761 1945 563532 730 749 TCATTCAACTGAAGAAAGGG 23 6743 6762 1946 563533 732 751 TTTCATTCAACTGAAGAAAG 8 6745 6764 1947 563534 733 752 ATTTCATTCAACTGAAGAAA 6 6746 6765 1948 563535 734 753 TATTTCATTCAACTGAAGAA 0 6747 6766 1949 563536 735 754 TTATTTCATTCAACTGAAGA 0 6748 6767 1950 563537 736 755 CTTATTTCATTCAACTGAAG 11 6749 6768 1951 337482 737 756 TCTTATTTCATTCAACTGAA 26 6750 6769 1952 563538 738 757 TTCTTATTTCATTCAACTGA 17 6751 6770 1953 563539 740 759 ATTTCTTATTTCATTCAACT 18 6753 6772 1954 563540 743 762 TACATTTCTTATTTCATTCA 20 6756 6775 1955 563541 767 786 TTCAGCAGGAATGCCATCAT 34 N/A N/A 1956 563542 768 787 ATTCAGCAGGAATGCCATCA 2 N/A N/A 1957 563543 769 788 CATTCAGCAGGAATGCCATC 21 N/A N/A 1958 563544 770 789 ACATTCAGCAGGAATGCCAT 5 N/A N/A 1959 563545 771 790 TACATTCAGCAGGAATGCCA 37 N/A N/A 1960 563546 772 791 GTACATTCAGCAGGAATGCC 50 7357 7376 1961 563547 773 792 GGTACATTCAGCAGGAATGC 64 7358 7377 76 563548 774 793 TGGTACATTCAGCAGGAATG 42 7359 7378 1962 563549 775 794 GTGGTACATTCAGCAGGAAT 51 7360 7379 1963 563550 776 795 GGTGGTACATTCAGCAGGAA 24 7361 7380 1964 563551 777 796 TGGTGGTACATTCAGCAGGA 47 7362 7381 1965 563552 778 797 ATGGTGGTACATTCAGCAGG 0 7363 7382 1966 563553 779 798 AATGGTGGTACATTCAGCAG 15 7364 7383 1967 563554 780 799 AAATGGTGGTACATTCAGCA 32 7365 7384 1968 563555 781 800 TAAATGGTGGTACATTCAGC 29 7366 7385 1969 563556 783 802 TATAAATGGTGGTACATTCA 33 7368 7387 1970 563557 784 803 TTATAAATGGTGGTACATTC 1 7369 7388 1971 563558 785 804 GTTATAAATGGTGGTACATT 4 7370 7389 1972 563559 786 805 TGTTATAAATGGTGGTACAT 0 7371 7390 1973 563560 787 806 CTGTTATAAATGGTGGTACA 4 7372 7391 1974 563561 788 807 TCTGTTATAAATGGTGGTAC 29 7373 7392 1975 337484 789 808 CTCTGTTATAAATGGTGGTA 62 7374 7393 74 563562 792 811 CACCTCTGTTATAAATGGTG 22 7377 7396 1976 563563 793 812 TCACCTCTGTTATAAATGGT 38 7378 7397 1977 337485 794 813 TTCACCTCTGTTATAAATGG 18 7379 7398 1978 563564 795 814 GTTCACCTCTGTTATAAATG 52 7380 7399 1979 563565 797 816 ATGTTCACCTCTGTTATAAA 24 7382 7401 1980 563566 798 817 TATGTTCACCTCTGTTATAA 2 7383 7402 1981 337486 799 818 GTATGTTCACCTCTGTTATA 32 7384 7403 1982 563567 800 819 TGTATGTTCACCTCTGTTAT 38 7385 7404 1983 337487 804 823 CACTTGTATGTTCACCTCTG 87 7389 7408 28 563568 1128 1147 TAATCGCAACTAGATGTAGC 39 9703 9722 1984 563569 1129 1148 GTAATCGCAACTAGATGTAG 26 9704 9723 1985 563570 1130 1149 AGTAATCGCAACTAGATGTA 17 9705 9724 1986 563571 1131 1150 CAGTAATCGCAACTAGATGT 43 9706 9725 1987 563572 1132 1151 CCAGTAATCGCAACTAGATG 39 9707 9726 1988 563573 1133 1152 GCCAGTAATCGCAACTAGAT 59 9708 9727 1989 563574 1134 1153 TGCCAGTAATCGCAACTAGA 57 9709 9728 1990 563575 1135 1154 TTGCCAGTAATCGCAACTAG 54 9710 9729 1991 563576 1136 1155 ATTGCCAGTAATCGCAACTA 43 9711 9730 1992 563577 1137 1156 CATTGCCAGTAATCGCAACT 49 9712 9731 1993 563578 1138 1157 ACATTGCCAGTAATCGCAAC 59 9713 9732 1994 563579 1139 1158 GACATTGCCAGTAATCGCAA 64 9714 9733 1995 563580 1140 1159 GGACATTGCCAGTAATCGCA 79 9715 9734 77 563581 1141 1160 GGGACATTGCCAGTAATCGC 47 9716 9735 1996 563582 1162 1181 TTGTTTTCCGGGATTGCATT 20 9737 9756 1997 563583 1163 1182 TTTGTTTTCCGGGATTGCAT 31 9738 9757 1998 563584 1167 1186 AATCTTTGTTTTCCGGGATT 14 9742 9761 1999 563585 1168 1187 AAATCTTTGTTTTCCGGGAT 54 9743 9762 2000 563586 1175 1194 AAACACCAAATCTTTGTTTT 32 9750 9769 2001 563587 1176 1195 AAAACACCAAATCTTTGTTT 7 9751 9770 2002 563588 1180 1199 GTAGAAAACACCAAATCTTT 18 9755 9774 2003 563589 1181 1200 AGTAGAAAACACCAAATCTT 0 9756 9775 2004 563590 1185 1204 CCCAAGTAGAAAACACCAAA 26 9760 9779 2005 563591 1186 1205 TCCCAAGTAGAAAACACCAA 27 9761 9780 2006 563592 1190 1209 GTGATCCCAAGTAGAAAACA 26 9765 9784 2007 563593 1191 1210 TGTGATCCCAAGTAGAAAAC 28 9766 9785 2008 563594 1192 1211 TTGTGATCCCAAGTAGAAAA 12 9767 9786 2009 563595 1193 1212 TTTGTGATCCCAAGTAGAAA 14 9768 9787 2010 563596 1200 1219 CTTTTGCTTTGTGATCCCAA 64 9775 9794 2011 563597 1204 1223 TGTCCTTTTGCTTTGTGATC 24 9779 9798 2012 563598 1205 1224 GTGTCCTTTTGCTTTGTGAT 31 9780 9799 2013 563599 1206 1225 AGTGTCCTTTTGCTTTGTGA 41 9781 9800 2014 563600 1210 1229 TTGAAGTGTCCTTTTGCTTT 21 9785 9804 2015 563601 1211 1230 GTTGAAGTGTCCTTTTGCTT 35 9786 9805 2016 563602 1212 1231 AGTTGAAGTGTCCTTTTGCT 27 9787 9806 2017 563603 1213 1232 CAGTTGAAGTGTCCTTTTGC 17 9788 9807 2018 563604 1214 1233 ACAGTTGAAGTGTCCTTTTG 0 9789 9808 2019 563605 1215 1234 GACAGTTGAAGTGTCCTTTT 19 9790 9809 2020 563606 1216 1235 GGACAGTTGAAGTGTCCTTT 34 9791 9810 2021 563607 1217 1236 TGGACAGTTGAAGTGTCCTT 12 9792 9811 2022 563608 1218 1237 CTGGACAGTTGAAGTGTCCT 39 9793 9812 2023 563609 1219 1238 TCTGGACAGTTGAAGTGTCC 10 9794 9813 2024 563610 1220 1239 CTCTGGACAGTTGAAGTGTC 6 9795 9814 2025 563611 1221 1240 CCTCTGGACAGTTGAAGTGT 24 9796 9815 2026 563612 1222 1241 CCCTCTGGACAGTTGAAGTG 24 9797 9816 2027 563613 1223 1242 ACCCTCTGGACAGTTGAAGT 31 9798 9817 2028 563614 1224 1243 AACCCTCTGGACAGTTGAAG 34 9799 9818 2029 563615 1225 1244 TAACCCTCTGGACAGTTGAA 34 9800 9819 2030 563616 1226 1245 ATAACCCTCTGGACAGTTGA 31 9801 9820 2031 563617 1227 1246 AATAACCCTCTGGACAGTTG 22 9802 9821 2032 563618 1228 1247 GAATAACCCTCTGGACAGTT 25 9803 9822 2033 563619 1229 1248 TGAATAACCCTCTGGACAGT 18 9804 9823 2034 563620 1230 1249 CTGAATAACCCTCTGGACAG 24 9805 9824 2035 563621 1231 1250 CCTGAATAACCCTCTGGACA 39 9806 9825 2036 563622 1232 1251 TCCTGAATAACCCTCTGGAC 31 N/A N/A 2037 563623 1233 1252 CTCCTGAATAACCCTCTGGA 15 N/A N/A 2038 563624 1234 1253 CCTCCTGAATAACCCTCTGG 27 N/A N/A 2039 563625 1235 1254 GCCTCCTGAATAACCCTCTG 25 N/A N/A 2040 563626 1236 1255 AGCCTCCTGAATAACCCTCT 32 N/A N/A 2041 563627 1237 1256 CAGCCTCCTGAATAACCCTC 44 N/A N/A 2042 563628 1238 1257 CCAGCCTCCTGAATAACCCT 26 N/A N/A 2043 563629 1239 1258 ACCAGCCTCCTGAATAACCC 23 N/A N/A 2044 337503 1240 1259 CACCAGCCTCCTGAATAACC 25 N/A N/A 2045 563630 1241 1260 CCACCAGCCTCCTGAATAAC 26 N/A N/A 2046 563631 1242 1261 ACCACCAGCCTCCTGAATAA 25 N/A N/A 2047 563632 1243 1262 CACCACCAGCCTCCTGAATA 33 N/A N/A 2048 563633 1244 1263 CCACCACCAGCCTCCTGAAT 45 N/A N/A 2049 563634 1248 1267 CATGCCACCACCAGCCTCCT 54 10220 10239 2050 563635 1250 1269 ATCATGCCACCACCAGCCTC 58 10222 10241 2051 563636 1251 1270 CATCATGCCACCACCAGCCT 61 10223 10242 2052 563637 1255 1274 CACTCATCATGCCACCACCA 68 10227 10246 78 563638 1256 1275 ACACTCATCATGCCACCACC 65 10228 10247 2053 563639 1260 1279 CTCCACACTCATCATGCCAC 76 10232 10251 79 563640 1262 1281 TTCTCCACACTCATCATGCC 55 10234 10253 2054 563641 1263 1282 TTTCTCCACACTCATCATGC 63 10235 10254 80 563642 1264 1283 TTTTCTCCACACTCATCATG 24 10236 10255 2055 563643 1265 1284 GTTTTCTCCACACTCATCAT 53 10237 10256 2056 563644 1857 1876 ATTTAAGAACTGTACAATTA 7 10829 10848 2057 563645 1858 1877 CATTTAAGAACTGTACAATT 15 10830 10849 2058 563646 1859 1878 ACATTTAAGAACTGTACAAT 4 10831 10850 2059 563647 1860 1879 AACATTTAAGAACTGTACAA 4 10832 10851 2060 563648 1861 1880 CAACATTTAAGAACTGTACA 4 10833 10852 2061 563649 1862 1881 ACAACATTTAAGAACTGTAC 22 10834 10853 2062 563650 1863 1882 TACAACATTTAAGAACTGTA 21 10835 10854 2063 563651 1864 1883 CTACAACATTTAAGAACTGT 44 10836 10855 2064 563652 1865 1884 ACTACAACATTTAAGAACTG 20 10837 10856 2065 563653 1866 1885 TACTACAACATTTAAGAACT 15 10838 10857 2066 563654 1867 1886 ATACTACAACATTTAAGAAC 17 10839 10858 2067 563655 1868 1887 AATACTACAACATTTAAGAA 11 10840 10859 2068 563656 1869 1888 TAATACTACAACATTTAAGA 9 10841 10860 2069 563657 1870 1889 TTAATACTACAACATTTAAG 3 10842 10861 2070 563658 1874 1893 GAAATTAATACTACAACATT 0 10846 10865 2071 563659 1878 1897 TTTTGAAATTAATACTACAA 0 10850 10869 2072 563660 1879 1898 GTTTTGAAATTAATACTACA 15 10851 10870 2073 563661 1880 1899 AGTTTTGAAATTAATACTAC 2 10852 10871 2074 563662 1881 1900 TAGTTTTGAAATTAATACTA 14 10853 10872 2075 563663 1882 1901 TTAGTTTTGAAATTAATACT 8 10854 10873 2076 563664 1888 1907 CGATTTTTAGTTTTGAAATT 0 10860 10879 2077 563665 1889 1908 ACGATTTTTAGTTTTGAAAT 0 10861 10880 2078 563666 1890 1909 GACGATTTTTAGTTTTGAAA 20 10862 10881 2079 563667 1891 1910 TGACGATTTTTAGTTTTGAA 17 10863 10882 2080 563668 1892 1911 CTGACGATTTTTAGTTTTGA 64 10864 10883 2081 563669 1893 1912 GCTGACGATTTTTAGTTTTG 66 10865 10884 81 563670 1894 1913 TGCTGACGATTTTTAGTTTT 45 10866 10885 2082 563671 1895 1914 GTGCTGACGATTTTTAGTTT 42 10867 10886 2083 563672 1896 1915 TGTGCTGACGATTTTTAGTT 50 10868 10887 2084 563673 1897 1916 CTGTGCTGACGATTTTTAGT 55 10869 10888 2085 563674 1898 1917 TCTGTGCTGACGATTTTTAG 53 10870 10889 2086 563675 1899 1918 CTCTGTGCTGACGATTTTTA 49 10871 10890 2087 563676 1900 1919 ACTCTGTGCTGACGATTTTT 22 10872 10891 2088 563677 1901 1920 TACTCTGTGCTGACGATTTT 8 10873 10892 2089 563678 1902 1921 ATACTCTGTGCTGACGATTT 61 10874 10893 2090 563679 1903 1922 CATACTCTGTGCTGACGATT 68 10875 10894 2091 563680 1904 1923 ACATACTCTGTGCTGACGAT 4 10876 10895 2092 563681 1905 1924 CACATACTCTGTGCTGACGA 73 10877 10896 82 563682 1909 1928 TTTACACATACTCTGTGCTG 67 10881 10900 83 563683 1911 1930 TTTTTACACATACTCTGTGC 58 10883 10902 2093 563684 1915 1934 CAGATTTTTACACATACTCT 54 10887 10906 2094 563685 1916 1935 ACAGATTTTTACACATACTC 52 10888 10907 2095 563686 1917 1936 TACAGATTTTTACACATACT 40 10889 10908 2096 563687 1918 1937 TTACAGATTTTTACACATAC 22 10890 10909 2097 337528 1920 1939 TATTACAGATTTTTACACAT 4 6720 6739 2098 563688 1922 1941 TGTATTACAGATTTTTACAC 0 10894 10913 2099 563689 1935 1954 CAGTTTAAAAATTTGTATTA 8 10907 10926 2100 563690 1938 1957 CATCAGTTTAAAAATTTGTA 18 10910 10929 2101 563691 1941 1960 AAGCATCAGTTTAAAAATTT 16 10913 10932 2102 563692 1942 1961 GAAGCATCAGTTTAAAAATT 16 10914 10933 2103 563693 1951 1970 TAGCAAAATGAAGCATCAGT 40 10923 10942 2104 563694 1952 1971 GTAGCAAAATGAAGCATCAG 42 10924 10943 2105 563695 1953 1972 TGTAGCAAAATGAAGCATCA 44 10925 10944 2106 563696 1954 1973 TTGTAGCAAAATGAAGCATC 48 10926 10945 2107 563697 1955 1974 TTTGTAGCAAAATGAAGCAT 19 10927 10946 2108 563698 1974 1993 AACATTTACTCCAAATTATT 27 10946 10965 2109 563699 1976 1995 CAAACATTTACTCCAAATTA 23 10948 10967 2110 563700 1978 1997 ATCAAACATTTACTCCAAAT 24 10950 10969 2111 563701 1981 2000 CATATCAAACATTTACTCCA 61 10953 10972 2112 563702 1982 2001 TCATATCAAACATTTACTCC 50 10954 10973 2113 563703 1983 2002 ATCATATCAAACATTTACTC 31 10955 10974 2114 563704 1990 2009 TAAATAAATCATATCAAACA 10 10962 10981 2115 563705 1993 2012 TCATAAATAAATCATATCAA 20 10965 10984 2116 563706 1994 2013 TTCATAAATAAATCATATCA 11 10966 10985 2117 563707 1995 2014 TTTCATAAATAAATCATATC 5 10967 10986 2118 563708 1996 2015 GTTTCATAAATAAATCATAT 0 10968 10987 2119 563709 1997 2016 GGTTTCATAAATAAATCATA 8 10969 10988 2120 563710 1998 2017 AGGTTTCATAAATAAATCAT 15 10970 10989 2121 563711 1999 2018 TAGGTTTCATAAATAAATCA 19 10971 10990 2122 563712 2001 2020 ATTAGGTTTCATAAATAAAT 12 10973 10992 2123 563713 2002 2021 CATTAGGTTTCATAAATAAA 2 10974 10993 2124 563714 2003 2022 TCATTAGGTTTCATAAATAA 7 10975 10994 2125 563715 2004 2023 TTCATTAGGTTTCATAAATA 11 10976 10995 2126 563716 2005 2024 CTTCATTAGGTTTCATAAAT 15 10977 10996 2127 563717 2006 2025 GCTTCATTAGGTTTCATAAA 49 10978 10997 2128 563718 2010 2029 TTCTGCTTCATTAGGTTTCA 57 10982 11001 2129 563719 2013 2032 TAATTCTGCTTCATTAGGTT 43 10985 11004 2130

TABLE 135 Inhibition of ANGPTL3 mRNA by 5-10-5 MOE gapmers targeting SEQ ID NO: 1 and 2 SEQ SEQ SEQ ID SEQ ID ID NO: NO: ID NO: NO: 2 SEQ ISIS 1 Start 1 Stop % 2 Start Stop ID NO Site Site Sequence inhibition Site Site NO 566915 343 362 TATGTAGTTCTTCTCAGTTC 22 3447 3466 2131 566916 350 369 TAGTTTATATGTAGTTCTTC 21 3454 3473 2132 566917 354 373 CTTGTAGTTTATATGTAGTT 12 3458 3477 2133 566918 358 377 TTGACTTGTAGTTTATATGT 12 3462 3481 2134 566919 360 379 TTTTGACTTGTAGTTTATAT 0 3464 3483 2135 566920 362 381 ATTTTTGACTTGTAGTTTAT 7 3466 3485 2136 566921 367 386 TCTTCATTTTTGACTTGTAG 33 3471 3490 2137 566922 371 390 TACCTCTTCATTTTTGACTT 22 3475 3494 2138 566923 377 396 ATTCTTTACCTCTTCATTTT 12 3481 3500 2139 566924 387 406 CAAGTGACATATTCTTTACC 36 3491 3510 2140 566925 389 408 TTCAAGTGACATATTCTTTA 31 3493 3512 2141 566926 394 413 TTGAGTTCAAGTGACATATT 18 3498 3517 2142 566927 396 415 AGTTGAGTTCAAGTGACATA 6 3500 3519 2143 566928 400 419 TTTGAGTTGAGTTCAAGTGA 11 3504 3523 2144 566929 408 427 TTTCAAGTTTTGAGTTGAGT 15 3512 3531 2145 566930 410 429 GCTTTCAAGTTTTGAGTTGA 13 3514 3533 2146 566931 412 431 AGGCTTTCAAGTTTTGAGTT 22 3516 3535 2147 566932 416 435 TAGGAGGCTTTCAAGTTTTG 4 3520 3539 2148 566933 419 438 TTCTAGGAGGCTTTCAAGTT 35 3523 3542 2149 566934 421 440 TCTTCTAGGAGGCTTTCAAG 26 3525 3544 2150 566935 429 448 GAATTTTTTCTTCTAGGAGG 1 3533 3552 2151 566936 434 453 AAGTAGAATTTTTTCTTCTA 0 3538 3557 2152 566937 436 455 TGAAGTAGAATTTTTTCTTC 11 3540 3559 2153 566938 438 457 GTTGAAGTAGAATTTTTTCT 29 3542 3561 2154 566939 441 460 TTTGTTGAAGTAGAATTTTT 11 3545 3564 2155 566940 443 462 TTTTTGTTGAAGTAGAATTT 35 3547 3566 2156 566941 464 483 TTGCTCTTCTAAATATTTCA 35 3568 3587 2157 566942 466 485 AGTTGCTCTTCTAAATATTT 53 3570 3589 2158 566943 468 487 TTAGTTGCTCTTCTAAATAT 18 3572 3591 2159 566944 471 490 TAGTTAGTTGCTCTTCTAAA 38 3575 3594 2160 566945 476 495 TAAGTTAGTTAGTTGCTCTT 28 3580 3599 2161 566946 478 497 ATTAAGTTAGTTAGTTGCTC 28 3582 3601 2162 566947 480 499 GAATTAAGTTAGTTAGTTGC 27 3584 3603 2163 566948 482 501 TTGAATTAAGTTAGTTAGTT 21 3586 3605 2164 566949 484 503 TTTTGAATTAAGTTAGTTAG 2 3588 3607 2165 566950 487 506 TGATTTTGAATTAAGTTAGT 9 3591 3610 2166 566951 490 509 GGTTGATTTTGAATTAAGTT 52 3594 3613 2167 566952 497 516 AGTTTCAGGTTGATTTTGAA 13 3601 3620 2168 566953 501 520 CTGGAGTTTCAGGTTGATTT 50 3605 3624 2169 566954 507 526 GGTGTTCTGGAGTTTCAGGT 35 3611 3630 2170 566955 509 528 TGGGTGTTCTGGAGTTTCAG 18 3613 3632 2171 566956 511 530 TCTGGGTGTTCTGGAGTTTC 32 3615 3634 2172 566957 513 532 CTTCTGGGTGTTCTGGAGTT 28 3617 3636 2173 566958 515 534 TACTTCTGGGTGTTCTGGAG 23 3619 3638 2174 566959 517 536 GTTACTTCTGGGTGTTCTGG 12 3621 3640 2175 566960 519 538 AAGTTACTTCTGGGTGTTCT 1 3623 3642 2176 566961 522 541 GTGAAGTTACTTCTGGGTGT 0 3626 3645 2177 566962 528 547 TTTTAAGTGAAGTTACTTCT 6 N/A N/A 2178 566963 530 549 AGTTTTAAGTGAAGTTACTT 16 N/A N/A 2179 566964 532 551 AAAGTTTTAAGTGAAGTTAC 12 N/A N/A 2180 566965 535 554 ACAAAAGTTTTAAGTGAAGT 8 N/A N/A 2181 337474 537 556 CTACAAAAGTTTTAAGTGAA 10 N/A N/A 2182 566966 539 558 TTCTACAAAAGTTTTAAGTG 46 N/A N/A 2183 566967 544 563 TGTTTTTCTACAAAAGTTTT 12 N/A N/A 2184 566968 546 565 CTTGTTTTTCTACAAAAGTT 0 N/A N/A 2185 566969 552 571 TATTATCTTGTTTTTCTACA 0 4290 4309 2186 566970 557 576 GATGCTATTATCTTGTTTTT 18 4295 4314 2187 566971 560 579 TTTGATGCTATTATCTTGTT 22 4298 4317 2188 566972 562 581 TCTTTGATGCTATTATCTTG 21 4300 4319 2189 566973 569 588 GAGAAGGTCTTTGATGCTAT 37 4307 4326 2190 566974 574 593 GTCTGGAGAAGGTCTTTGAT 26 4312 4331 2191 566975 576 595 CGGTCTGGAGAAGGTCTTTG 20 4314 4333 2192 566976 578 597 CACGGTCTGGAGAAGGTCTT 53 4316 4335 2193 566977 580 599 TCCACGGTCTGGAGAAGGTC 58 4318 4337 2194 566978 582 601 CTTCCACGGTCTGGAGAAGG 39 4320 4339 2195 566979 584 603 GTCTTCCACGGTCTGGAGAA 63 4322 4341 2196 566980 586 605 TGGTCTTCCACGGTCTGGAG 81 4324 4343 2197 566981 588 607 ATTGGTCTTCCACGGTCTGG 57 4326 4345 2198 566982 590 609 ATATTGGTCTTCCACGGTCT 60 4328 4347 2199 566983 592 611 TTATATTGGTCTTCCACGGT 49 4330 4349 2200 566984 594 613 GTTTATATTGGTCTTCCACG 54 4332 4351 2201 566985 596 615 TTGTTTATATTGGTCTTCCA 36 4334 4353 2202 566986 598 617 AATTGTTTATATTGGTCTTC 23 4336 4355 2203 566987 600 619 TTAATTGTTTATATTGGTCT 26 4338 4357 2204 566988 602 621 GTTTAATTGTTTATATTGGT 23 4340 4359 2205 566989 604 623 TGGTTTAATTGTTTATATTG 8 4342 4361 2206 566990 606 625 GTTGGTTTAATTGTTTATAT 1 4344 4363 2207 544120 707 726 AGTTCTTGGTGCTCTTGGCT 78 6720 6739 15 337487 804 823 CACTTGTATGTTCACCTCTG 82 7389 7408 28 566991 912 931 TTTGTGATCCATCTATTCGA 25 7899 7918 2208 566992 913 932 TTTTGTGATCCATCTATTCG 12 7900 7919 2209 566993 920 939 ATTGAAGTTTTGTGATCCAT 32 7907 7926 2210 566994 921 940 CATTGAAGTTTTGTGATCCA 26 7908 7927 2211 566995 922 941 TCATTGAAGTTTTGTGATCC 0 7909 7928 2212 566996 923 942 TTCATTGAAGTTTTGTGATC 1 7910 7929 2213 566997 924 943 TTTCATTGAAGTTTTGTGAT 20 7911 7930 2214 566998 944 963 ATATTTGTAGTTCTCCCACG 35 7931 7950 2215 566999 952 971 CCAAAACCATATTTGTAGTT 13 7939 7958 2216 567000 953 972 CCCAAAACCATATTTGTAGT 21 7940 7959 2217 567001 954 973 TCCCAAAACCATATTTGTAG 0 7941 7960 2218 567002 955 974 CTCCCAAAACCATATTTGTA 5 7942 7961 2219 567003 958 977 AGCCTCCCAAAACCATATTT 0 7945 7964 2220 567004 960 979 CAAGCCTCCCAAAACCATAT 14 7947 7966 2221 567005 961 980 TCAAGCCTCCCAAAACCATA 0 7948 7967 2222 567006 962 981 ATCAAGCCTCCCAAAACCAT 17 7949 7968 2223 567007 963 982 CATCAAGCCTCCCAAAACCA 31 7950 7969 2224 567008 964 983 CCATCAAGCCTCCCAAAACC 11 7951 7970 2225 567009 965 984 TCCATCAAGCCTCCCAAAAC 27 N/A N/A 2226 567010 966 985 CTCCATCAAGCCTCCCAAAA 42 N/A N/A 2227 567011 972 991 AAAATTCTCCATCAAGCCTC 48 N/A N/A 2228 567012 974 993 CCAAAATTCTCCATCAAGCC 41 N/A N/A 2229 567013 975 994 ACCAAAATTCTCCATCAAGC 49 N/A N/A 2230 567014 978 997 CCAACCAAAATTCTCCATCA 32 N/A N/A 2231 567015 979 998 CCCAACCAAAATTCTCCATC 47 N/A N/A 2232 337497 980 999 GCCCAACCAAAATTCTCCAT 46 N/A N/A 2233 567016 981 1000 GGCCCAACCAAAATTCTCCA 48 N/A N/A 2234 567017 982 1001 AGGCCCAACCAAAATTCTCC 30 9557 9576 2235 567018 983 1002 TAGGCCCAACCAAAATTCTC 0 9558 9577 2236 567019 984 1003 CTAGGCCCAACCAAAATTCT 31 9559 9578 2237 567020 985 1004 TCTAGGCCCAACCAAAATTC 39 9560 9579 2238 233721 986 1005 CTCTAGGCCCAACCAAAATT 15 9561 9580 2239 567021 987 1006 TCTCTAGGCCCAACCAAAAT 36 9562 9581 2240 567022 988 1007 TTCTCTAGGCCCAACCAAAA 26 9563 9582 2241 567023 989 1008 CTTCTCTAGGCCCAACCAAA 44 9564 9583 2242 567024 993 1012 ATATCTTCTCTAGGCCCAAC 29 9568 9587 2243 567025 994 1013 TATATCTTCTCTAGGCCCAA 41 9569 9588 2244 567026 995 1014 GTATATCTTCTCTAGGCCCA 53 9570 9589 2245 567027 1000 1019 ATGGAGTATATCTTCTCTAG 18 9575 9594 2246 567028 1004 1023 CACTATGGAGTATATCTTCT 35 9579 9598 2247 567029 1005 1024 TCACTATGGAGTATATCTTC 9 9580 9599 2248 567030 1006 1025 TTCACTATGGAGTATATCTT 11 9581 9600 2249 567031 1010 1029 TTGCTTCACTATGGAGTATA 43 9585 9604 2250 567032 1011 1030 ATTGCTTCACTATGGAGTAT 4 9586 9605 2251 567033 1015 1034 TTAGATTGCTTCACTATGGA 17 9590 9609 2252 567034 1016 1035 ATTAGATTGCTTCACTATGG 35 9591 9610 2253 567035 1017 1036 AATTAGATTGCTTCACTATG 18 9592 9611 2254 567036 1018 1037 TAATTAGATTGCTTCACTAT 17 9593 9612 2255 567037 1019 1038 ATAATTAGATTGCTTCACTA 19 9594 9613 2256 567038 1020 1039 CATAATTAGATTGCTTCACT 27 9595 9614 2257 567039 1021 1040 ACATAATTAGATTGCTTCAC 17 9596 9615 2258 337498 1022 1041 AACATAATTAGATTGCTTCA 9 9597 9616 2259 567040 1023 1042 AAACATAATTAGATTGCTTC 0 9598 9617 2260 567041 1024 1043 AAAACATAATTAGATTGCTT 0 9599 9618 2261 567042 1025 1044 TAAAACATAATTAGATTGCT 23 9600 9619 2262 567043 1026 1045 GTAAAACATAATTAGATTGC 25 9601 9620 2263 567044 1027 1046 CGTAAAACATAATTAGATTG 0 9602 9621 2264 567045 1048 1067 TTCCAGTCTTCCAACTCAAT 9 9623 9642 2265 337500 1050 1069 CTTTCCAGTCTTCCAACTCA 30 9625 9644 2266 567046 1057 1076 TTGTTGTCTTTCCAGTCTTC 40 9632 9651 2267 567047 1064 1083 ATAATGTTTGTTGTCTTTCC 26 9639 9658 2268 567048 1065 1084 TATAATGTTTGTTGTCTTTC 6 9640 9659 2269 567049 1066 1085 ATATAATGTTTGTTGTCTTT 9 9641 9660 2270 567050 1069 1088 TCAATATAATGTTTGTTGTC 20 9644 9663 2271 567051 1073 1092 ATATTCAATATAATGTTTGT 15 9648 9667 2272 567052 1074 1093 AATATTCAATATAATGTTTG 16 9649 9668 2273 567053 1075 1094 GAATATTCAATATAATGTTT 7 9650 9669 2274 567054 1076 1095 AGAATATTCAATATAATGTT 3 9651 9670 2275 567055 1077 1096 AAGAATATTCAATATAATGT 7 9652 9671 2276 567056 1085 1104 CAAGTAAAAAGAATATTCAA 0 9660 9679 2277 567057 1086 1105 CCAAGTAAAAAGAATATTCA 0 9661 9680 2278 567058 1087 1106 CCCAAGTAAAAAGAATATTC 13 9662 9681 2279 567059 1090 1109 TTTCCCAAGTAAAAAGAATA 0 9665 9684 2280 567060 1091 1110 ATTTCCCAAGTAAAAAGAAT 2 9666 9685 2281 567061 1092 1111 GATTTCCCAAGTAAAAAGAA 14 9667 9686 2282 567062 1093 1112 TGATTTCCCAAGTAAAAAGA 14 9668 9687 2283 567063 1127 1146 AATCGCAACTAGATGTAGCG 15 9702 9721 2284 563874 1586 1605 ATTCTTTAAGGTTATGTGAT 13 10558 10577 2285 563875 1587 1606 TATTCTTTAAGGTTATGTGA 25 10559 10578 2286 563876 1591 1610 ACGGTATTCTTTAAGGTTAT 50 10563 10582 2287 563877 1592 1611 AACGGTATTCTTTAAGGTTA 48 10564 10583 2288 563878 1593 1612 AAACGGTATTCTTTAAGGTT 45 10565 10584 2289 563879 1594 1613 TAAACGGTATTCTTTAAGGT 16 10566 10585 2290 563880 1595 1614 GTAAACGGTATTCTTTAAGG 14 10567 10586 2291 563881 1596 1615 TGTAAACGGTATTCTTTAAG 0 10568 10587 2292 563882 1597 1616 ATGTAAACGGTATTCTTTAA 10 10569 10588 2293 563883 1598 1617 AATGTAAACGGTATTCTTTA 12 10570 10589 2294 563884 1599 1618 AAATGTAAACGGTATTCTTT 15 10571 10590 2295 563885 1600 1619 GAAATGTAAACGGTATTCTT 13 10572 10591 2296 563886 1601 1620 AGAAATGTAAACGGTATTCT 22 10573 10592 2297 563887 1602 1621 GAGAAATGTAAACGGTATTC 35 10574 10593 2298 563888 1603 1622 TGAGAAATGTAAACGGTATT 14 10575 10594 2299 563889 1604 1623 TTGAGAAATGTAAACGGTAT 0 10576 10595 2300 563890 1605 1624 ATTGAGAAATGTAAACGGTA 18 10577 10596 2301 563891 1606 1625 GATTGAGAAATGTAAACGGT 40 10578 10597 2302 563892 1607 1626 TGATTGAGAAATGTAAACGG 33 10579 10598 2303 563893 1608 1627 TTGATTGAGAAATGTAAACG 7 10580 10599 2304 563894 1609 1628 TTTGATTGAGAAATGTAAAC 0 10581 10600 2305 563895 1610 1629 TTTTGATTGAGAAATGTAAA 0 10582 10601 2306 563896 1611 1630 ATTTTGATTGAGAAATGTAA 0 10583 10602 2307 563897 1612 1631 AATTTTGATTGAGAAATGTA 0 10584 10603 2308 563898 1613 1632 GAATTTTGATTGAGAAATGT 4 10585 10604 2309 563899 1614 1633 AGAATTTTGATTGAGAAATG 4 10586 10605 2310 563900 1615 1634 AAGAATTTTGATTGAGAAAT 26 10587 10606 2311 563901 1617 1636 ATAAGAATTTTGATTGAGAA 4 10589 10608 2312 563902 1618 1637 TATAAGAATTTTGATTGAGA 0 10590 10609 2313 563903 1619 1638 TTATAAGAATTTTGATTGAG 0 10591 10610 2314 563904 1620 1639 ATTATAAGAATTTTGATTGA 0 10592 10611 2315 563905 1621 1640 TATTATAAGAATTTTGATTG 3 10593 10612 2316 563906 1622 1641 GTATTATAAGAATTTTGATT 1 10594 10613 2317 563907 1623 1642 AGTATTATAAGAATTTTGAT 44 10595 10614 2318 563908 1624 1643 TAGTATTATAAGAATTTTGA 29 10596 10615 2319 563909 1632 1651 AAAACAAATAGTATTATAAG 11 10604 10623 2320 563910 1633 1652 TAAAACAAATAGTATTATAA 16 10605 10624 2321 563911 1652 1671 ATTCCCACATCACAAAATTT 27 10624 10643 2322 563912 1653 1672 GATTCCCACATCACAAAATT 21 10625 10644 2323 563913 1654 1673 TGATTCCCACATCACAAAAT 49 10626 10645 2324 563914 1658 1677 AAATTGATTCCCACATCACA 47 10630 10649 2325 563915 1659 1678 AAAATTGATTCCCACATCAC 48 10631 10650 2326 563916 1663 1682 ATCTAAAATTGATTCCCACA 58 10635 10654 2327 563917 1667 1686 GACCATCTAAAATTGATTCC 41 10639 10658 2328 563918 1668 1687 TGACCATCTAAAATTGATTC 25 10640 10659 2329 563919 1669 1688 GTGACCATCTAAAATTGATT 33 10641 10660 2330 563920 1670 1689 TGTGACCATCTAAAATTGAT 34 10642 10661 2331 563921 1671 1690 TTGTGACCATCTAAAATTGA 20 10643 10662 2332 563922 1672 1691 ATTGTGACCATCTAAAATTG 2 10644 10663 2333 563923 1673 1692 GATTGTGACCATCTAAAATT 43 10645 10664 2334 563924 1674 1693 AGATTGTGACCATCTAAAAT 39 10646 10665 2335 563925 1675 1694 TAGATTGTGACCATCTAAAA 36 10647 10666 2336 563926 1676 1695 CTAGATTGTGACCATCTAAA 56 10648 10667 2337 563927 1677 1696 TCTAGATTGTGACCATCTAA 37 10649 10668 2338 563928 1678 1697 ATCTAGATTGTGACCATCTA 46 10650 10669 2339 563929 1679 1698 AATCTAGATTGTGACCATCT 56 10651 10670 2340 563930 1680 1699 TAATCTAGATTGTGACCATC 46 10652 10671 2341 563931 1681 1700 ATAATCTAGATTGTGACCAT 35 10653 10672 2342 563932 1682 1701 TATAATCTAGATTGTGACCA 45 10654 10673 2343 563933 1683 1702 TTATAATCTAGATTGTGACC 37 10655 10674 2344 563934 1686 1705 TGATTATAATCTAGATTGTG 28 10658 10677 2345 563935 1687 1706 TTGATTATAATCTAGATTGT 0 10659 10678 2346 563936 1688 1707 ATTGATTATAATCTAGATTG 0 10660 10679 2347 563937 1689 1708 TATTGATTATAATCTAGATT 0 10661 10680 2348 563938 1690 1709 CTATTGATTATAATCTAGAT 5 10662 10681 2349 563939 1691 1710 CCTATTGATTATAATCTAGA 0 10663 10682 2350 563940 1692 1711 ACCTATTGATTATAATCTAG 9 10664 10683 2351 563941 1693 1712 CACCTATTGATTATAATCTA 5 10665 10684 2352 563942 1694 1713 TCACCTATTGATTATAATCT 0 10666 10685 2353 563943 1695 1714 TTCACCTATTGATTATAATC 10 10667 10686 2354 563944 1696 1715 GTTCACCTATTGATTATAAT 31 10668 10687 2355 563945 1697 1716 AGTTCACCTATTGATTATAA 15 10669 10688 2356 563946 1698 1717 AAGTTCACCTATTGATTATA 31 10670 10689 2357 563947 1700 1719 ATAAGTTCACCTATTGATTA 9 10672 10691 2358 563948 1701 1720 AATAAGTTCACCTATTGATT 5 10673 10692 2359 563949 1702 1721 TAATAAGTTCACCTATTGAT 14 10674 10693 2360 563950 1703 1722 TTAATAAGTTCACCTATTGA 0 10675 10694 2361

TABLE 136 Inhibition of ANGPTL3 mRNA by 5-10-5 MOE gapmers targeting SEQ ID NO: 1 and 2 SEQ SEQ SEQ ID SEQ ID ID NO: NO: ID NO: NO: 2 SEQ ISIS 1 Start 1 Stop % 2 Start Stop ID NO Site Site Sequence inhibition Site Site NO 567064 N/A N/A TGAGTATTCTCGACTTACCT 26 8770 8789 2362 567065 N/A N/A AAGTGAGTATTCTCGACTTA 2 8773 8792 2363 567066 N/A N/A ATTAAGTGAGTATTCTCGAC 20 8776 8795 2364 567067 N/A N/A CCAGAATTAAGTGAGTATTC 36 8781 8800 2365 567068 N/A N/A GCTTTCTTACCAGAATTAAG 75 8790 8809 84 567069 N/A N/A GTTGCTTTCTTACCAGAATT 78 8793 8812 85 567070 N/A N/A TGGGTTGCTTTCTTACCAGA 26 8796 8815 2366 567071 N/A N/A AAATGGGTTGCTTTCTTACC 3 8799 8818 2367 567072 N/A N/A TACAAATGGGTTGCTTTCTT 24 8802 8821 2368 567073 N/A N/A AAGTACAAATGGGTTGCTTT 24 8805 8824 2369 567074 N/A N/A GTAAATACAAGTACAAATGG 7 8813 8832 2370 567075 N/A N/A TTGCTGGTAAATACAAGTAC 24 8819 8838 2371 567076 N/A N/A TAAGGATTGCTGGTAAATAC 6 8825 8844 2372 567077 N/A N/A TTTTAAGGATTGCTGGTAAA 4 8828 8847 2373 567078 N/A N/A GCTTCATTTTAAGGATTGCT 60 8834 8853 87 567079 N/A N/A GAAGCTTCATTTTAAGGATT 0 8837 8856 2374 567080 N/A N/A TAGGAAGCTTCATTTTAAGG 9 8840 8859 2375 567081 N/A N/A TAGTAGGAAGCTTCATTTTA 18 8843 8862 2376 567082 N/A N/A TTGAGTTAGTAGGAAGCTTC 30 8849 8868 2377 567083 N/A N/A ATTGCTATTGAGTTAGTAGG 21 8856 8875 2378 567084 N/A N/A CTTATTGCTATTGAGTTAGT 28 8859 8878 2379 567085 N/A N/A ATTGTCTTATTGCTATTGAG 16 8864 8883 2380 567086 N/A N/A ACTATTGTCTTATTGCTATT 10 8867 8886 2381 567087 N/A N/A TTCACTATTGTCTTATTGCT 35 8870 8889 2382 567088 N/A N/A ACATTCACTATTGTCTTATT 30 8873 8892 2383 567089 N/A N/A TAAACATTCACTATTGTCTT 58 8876 8895 2384 567090 N/A N/A CATTAAACATTCACTATTGT 28 8879 8898 2385 567091 N/A N/A GTTTTCATTAAACATTCACT 54 8884 8903 2386 567092 N/A N/A AAATACTGTTTTCATTAAAC 34 8891 8910 2387 567093 N/A N/A AAAGTATTTATAAAATACTG 0 8903 8922 2388 567094 N/A N/A CCTTTTTATTAAAGTATTTA 0 8913 8932 2389 567095 N/A N/A CAATCCTTTTTATTAAAGTA 10 8917 8936 2390 567096 N/A N/A CTTCATCACAATCCTTTTTA 52 8925 8944 2391 567097 N/A N/A GTTCTTCATCACAATCCTTT 57 8928 8947 2392 567098 N/A N/A ATTGTTCTTCATCACAATCC 37 8931 8950 2393 567099 N/A N/A TAGATTGTTCTTCATCACAA 31 8934 8953 2394 567100 N/A N/A AAATAGATTGTTCTTCATCA 11 8937 8956 2395 567101 N/A N/A AACAAATATAAATAGATTGT 0 8946 8965 2396 567102 N/A N/A CAAATAACAAATATAAATAG 3 8951 8970 2397 567103 N/A N/A TGGAATTAAAAACAAATAAC 3 8963 8982 2398 567104 N/A N/A TTATTGGAATTAAAAACAAA 12 8967 8986 2399 567105 N/A N/A TTTTTATTGGAATTAAAAAC 17 8970 8989 2400 567106 N/A N/A TAATAACTTTTTTCTGTAAT 6 9001 9020 2401 567107 N/A N/A GTTCTTAATAACTTTTTTCT 21 9006 9025 2402 567108 N/A N/A AAAAGCATGGTTCTTAATAA 0 9015 9034 2403 567109 N/A N/A AAATTTAAAAGCATGGTTCT 0 9021 9040 2404 567110 N/A N/A AGGAATAAATTTAAAAAATC 0 9046 9065 2405 567111 N/A N/A AGACAGGAATAAATTTAAAA 7 9050 9069 2406 567112 N/A N/A AAAAGACAGGAATAAATTTA 0 9053 9072 2407 567113 N/A N/A CTTTCTTTGTAGAAAAAGAC 29 9066 9085 2408 567114 N/A N/A ATGCTTTCTTTGTAGAAAAA 12 9069 9088 2409 567115 N/A N/A GCTTAATGTATGCTTTCTTT 67 9078 9097 88 567116 N/A N/A TTTGCTTAATGTATGCTTTC 21 9081 9100 2410 567117 N/A N/A GTATTTGCTTAATGTATGCT 0 9084 9103 2411 567118 N/A N/A TTGGTATTTGCTTAATGTAT 0 9087 9106 2412 567119 N/A N/A CCTTTGGTATTTGCTTAATG 35 9090 9109 2413 567120 N/A N/A TGGCCTTTGGTATTTGCTTA 0 9093 9112 2414 567121 N/A N/A TAAACCTGGCCTTTGGTATT 27 9099 9118 2415 567122 N/A N/A ATGTAAACCTGGCCTTTGGT 16 9102 9121 2416 567123 N/A N/A CAAATGTAAACCTGGCCTTT 0 9105 9124 2417 567124 N/A N/A CTTCAAATGTAAACCTGGCC 25 9108 9127 2418 567125 N/A N/A TTTCTTCAAATGTAAACCTG 2 9111 9130 2419 567126 N/A N/A TGTCACTTTCTTCAAATGTA 57 9117 9136 2420 567127 N/A N/A TAATGTCACTTTCTTCAAAT 6 9120 9139 2421 567128 N/A N/A AATAATAATGTCACTTTCTT 3 9125 9144 2422 567129 N/A N/A GAGTAATAATAATGTCACTT 18 9129 9148 2423 567130 N/A N/A GACTTGAGTAATAATAATGT 1 9134 9153 2424 567131 N/A N/A CCTAGAGACTTGAGTAATAA 32 9140 9159 2425 567132 N/A N/A ATTCCTAGAGACTTGAGTAA 8 9143 9162 2426 567133 N/A N/A AAGTATTCCTAGAGACTTGA 11 9147 9166 2427 567134 N/A N/A GTTAAGTATTCCTAGAGACT 61 9150 9169 89 567135 N/A N/A TGTGTTAAGTATTCCTAGAG 28 9153 9172 2428 567136 N/A N/A AGAGATGTGTTAAGTATTCC 31 9158 9177 2429 567137 N/A N/A GTCAAGAGATGTGTTAAGTA 52 9162 9181 2430 567138 N/A N/A ACAGTCAAGAGATGTGTTAA 22 9165 9184 2431 567139 N/A N/A TATACAGTCAAGAGATGTGT 30 9168 9187 2432 567140 N/A N/A CCATATACAGTCAAGAGATG 45 9171 9190 2433 567141 N/A N/A GTAAGTTGAACTAACTACTG 9 7497 7516 2434 567142 N/A N/A TGAGTAAGTTGAACTAACTA 0 7500 7519 2435 567143 N/A N/A TAATGAGTAAGTTGAACTAA 2 7503 7522 2436 567144 N/A N/A AGGTTAATCTTCCTAATACG 18 7523 7542 2437 567145 N/A N/A ATAACCAGGTTAATCTTCCT 34 7529 7548 2438 567146 N/A N/A ATGATAACCAGGTTAATCTT 13 7532 7551 2439 567147 N/A N/A AACAATGATAACCAGGTTAA 7 7536 7555 2440 567148 N/A N/A TAAAACAATGATAACCAGGT 45 7539 7558 2441 567149 N/A N/A GTATAAAACAATGATAACCA 26 7542 7561 2442 567150 N/A N/A CGAATACTCATATATATTTC 25 7572 7591 2443 567151 N/A N/A ATACGAATACTCATATATAT 30 7575 7594 2444 567152 N/A N/A TTTATACGAATACTCATATA 32 7578 7597 2445 567153 N/A N/A ATATTTATACGAATACTCAT 25 7581 7600 2446 567154 N/A N/A GTATTATATTTATACGAATA 0 7586 7605 2447 567155 N/A N/A AAAAGTATTATATTTATACG 0 7590 7609 2448 567156 N/A N/A GGTAAAAGTATTATATTTAT 0 7593 7612 2449 567157 N/A N/A ACAAGGTAAAAGTATTATAT 10 7597 7616 2450 567158 N/A N/A TAAACAAGGTAAAAGTATTA 11 7600 7619 2451 567159 N/A N/A ACATAAACAAGGTAAAAGTA 3 7603 7622 2452 567160 N/A N/A TTGAGTAAATACATAAACAA 12 7613 7632 2453 567161 N/A N/A GAGAATATTGAGTAAATACA 4 7620 7639 2454 567162 N/A N/A AAGGAGAATATTGAGTAAAT 8 7623 7642 2455 567163 N/A N/A GAAAAGGAGAATATTGAGTA 3 7626 7645 2456 567164 N/A N/A GAGGAAAAGGAGAATATTGA 19 7629 7648 2457 567165 N/A N/A TTAGAGGAAAAGGAGAATAT 41 7632 7651 2458 567166 N/A N/A ATTATTTTAGAGGAAAAGGA 30 7638 7657 2459 567167 N/A N/A CAGATTATTTTAGAGGAAAA 9 7641 7660 2460 567168 N/A N/A CTTCAGATTATTTTAGAGGA 24 7644 7663 2461 567169 N/A N/A TAGTCACTTCAGATTATTTT 38 7650 7669 2462 567170 N/A N/A TAATAGTCACTTCAGATTAT 13 7653 7672 2463 567171 N/A N/A TGATAATAGTCACTTCAGAT 39 7656 7675 2464 567172 N/A N/A TATTGATAATAGTCACTTCA 41 7659 7678 2465 567173 N/A N/A ACTTATTGATAATAGTCACT 29 7662 7681 2466 567174 N/A N/A TAAACTTATTGATAATAGTC 14 7665 7684 2467 567175 N/A N/A TAGTAAACTTATTGATAATA 31 7668 7687 2468 567176 N/A N/A GCATAGTAAACTTATTGATA 23 7671 7690 2469 567177 N/A N/A TTGGCATAGTAAACTTATTG 21 7674 7693 2470 567178 N/A N/A ATTTTGGCATAGTAAACTTA 8 7677 7696 2471 567179 N/A N/A TGAATTTTGGCATAGTAAAC 5 7680 7699 2472 567180 N/A N/A TTAATGAATTTTGGCATAGT 0 7684 7703 2473 567181 N/A N/A CAATTAATGAATTTTGGCAT 39 7687 7706 2474 567182 N/A N/A AAAGGCAATTAATGAATTTT 12 7692 7711 2475 567183 N/A N/A GTGAAAGGCAATTAATGAAT 28 7695 7714 2476 567184 N/A N/A TTAAGTGAAAGGCAATTAAT 7 7699 7718 2477 567185 N/A N/A AAGTTAAGTGAAAGGCAATT 25 7702 7721 2478 567186 N/A N/A CCAAAAGTTAAGTGAAAGGC 50 7706 7725 2479 567187 N/A N/A GTCCCAAAAGTTAAGTGAAA 30 7709 7728 2480 567188 N/A N/A ATGGTCCCAAAAGTTAAGTG 39 7712 7731 2481 567189 N/A N/A ATTATGGTCCCAAAAGTTAA 19 7715 7734 2482 567190 N/A N/A TTTATTATGGTCCCAAAAGT 33 7718 7737 2483 567191 N/A N/A TTATTATTTATTATGGTCCC 50 7724 7743 2484 567192 N/A N/A ATGGCAATACATTTTATTAT 13 7737 7756 2485 567193 N/A N/A GTTATGGCAATACATTTTAT 39 7740 7759 2486 567194 N/A N/A TAATGTTATGGCAATACATT 0 7744 7763 2487 567195 N/A N/A TATTAATGTTATGGCAATAC 22 7747 7766 2488 567196 N/A N/A GTTTATTAATGTTATGGCAA 28 7750 7769 2489 567197 N/A N/A GTAGTTTATTAATGTTATGG 20 7753 7772 2490 567198 N/A N/A AAGGTAGTTTATTAATGTTA 27 7756 7775 2491 567199 N/A N/A TGTAAGGTAGTTTATTAATG 0 7759 7778 2492 567200 N/A N/A TTTTGTAAGGTAGTTTATTA 0 7762 7781 2493 567201 N/A N/A TGGTTTTGTAAGGTAGTTTA 18 7765 7784 2494 567202 N/A N/A TGGTGGTTTTGTAAGGTAGT 0 7768 7787 2495 567203 N/A N/A AATTGGTGGTTTTGTAAGGT 11 7771 7790 2496 567204 N/A N/A TTTAATTGGTGGTTTTGTAA 0 7774 7793 2497 567205 N/A N/A TTGATTTTAATTGGTGGTTT 19 7779 7798 2498 567206 N/A N/A TGTTTGATTTTAATTGGTGG 26 7782 7801 2499 567207 N/A N/A ATGTAAATAACACTTTTTTG 1 7804 7823 2500 567208 N/A N/A CAGATGTAAATAACACTTTT 1 7807 7826 2501 567209 N/A N/A TGACAGATGTAAATAACACT 21 7810 7829 2502 567210 N/A N/A ATGTTGACAGATGTAAATAA 0 7814 7833 2503 567211 N/A N/A TTTATGTTGACAGATGTAAA 0 7817 7836 2504 567212 N/A N/A AGATTTATGTTGACAGATGT 0 7820 7839 2505 567213 N/A N/A AGTAGATTTATGTTGACAGA 19 7823 7842 2506 567214 N/A N/A TTTAGTAGATTTATGTTGAC 4 7826 7845 2507 567215 N/A N/A ATTTTTAGTAGATTTATGTT 0 7829 7848 2508 567216 N/A N/A CATGTATTTTTAGTAGATTT 5 7834 7853 2509 567217 N/A N/A GAAATCATGTATTTTTAGTA 0 7839 7858 2510 567218 N/A N/A ATTGTATTTGATGGATATCT 43 6875 6894 2511 567219 N/A N/A GATACATTGTATTTGATGGA 20 6880 6899 2512 567220 N/A N/A TAGGTTGATACATTGTATTT 18 6886 6905 2513 567221 N/A N/A CAGTTTAGGTTGATACATTG 18 6891 6910 2514 567222 N/A N/A GCATCCAGTTTAGGTTGATA 31 6896 6915 2515 567223 N/A N/A CCCCAGCATCCAGTTTAGGT 14 6901 6920 2516 567224 N/A N/A AAGAACCCCAGCATCCAGTT 41 6906 6925 2517 567225 N/A N/A GTGTAAAAAGAACCCCAGCA 0 6913 6932 2518 567226 N/A N/A ATAGGGTGTAAAAAGAACCC 13 6918 6937 2519 567227 N/A N/A CTTTTATAGGGTGTAAAAAG 0 6923 6942 2520 567228 N/A N/A TATGTCTTTTATAGGGTGTA 26 6928 6947 2521 567229 N/A N/A TTAGGTATGTCTTTTATAGG 0 6933 6952 2522 567230 N/A N/A TTGTCTTAGGTATGTCTTTT 30 6938 6957 2523 567231 N/A N/A CTCTGATTGTCTTAGGTATG 27 6944 6963 2524 567232 N/A N/A TATTTCTCTGATTGTCTTAG 21 6949 6968 2525 567233 N/A N/A TCCATATTTGTATTTCTCTG 61 6959 6978 90 567234 N/A N/A TCAAGTCCATATTTGTATTT 20 6964 6983 2526 567235 N/A N/A AATAATCAAGTCCATATTTG 0 6969 6988 2527 567236 N/A N/A TTATCTAATAATCAAGTCCA 0 6975 6994 2528 567237 N/A N/A CTATATTATCTAATAATCAA 12 6980 6999 2529 567238 N/A N/A TAAACCTTCTATATTATCTA 12 6988 7007 2530 567239 N/A N/A AATTAATAAACCTTCTATAT 0 6994 7013 2531 567240 N/A N/A TAAGTACAGGTTGGACACTG 0 9504 9523 2532 567241 N/A N/A GTTATTAAGTACAGGTTGGA 2 9509 9528 2533 567242 N/A N/A TGTGAGTTATTAAGTACAGG 0 9514 9533 2534 567243 N/A N/A AAATCTGTGAGTTATTAAGT 0 9519 9538 2535 567244 N/A N/A GTTTTAAAAATCTGTGAGTT 19 9526 9545 2536 567245 N/A N/A CAAAATTCTCCTGAAAAGAA 20 9548 9567 2537 567246 N/A N/A CCCAACCAAAATTCTCCTGA 48 9554 9573 2538 567247 N/A N/A ACCTGAATAACCCTCTGGAC 21 9807 9826 2539 567248 N/A N/A AAGATACCTGAATAACCCTC 30 9812 9831 2540 567249 N/A N/A AGAAAAAGATACCTGAATAA 0 9817 9836 2541 567250 N/A N/A TGGTATCAGAAAAAGATACC 0 9824 9843 2542 567251 N/A N/A AGTATTGGTATCAGAAAAAG 0 9829 9848 2543 567252 N/A N/A AATAAAGTATTGGTATCAGA 10 9834 9853 2544 567253 N/A N/A ATGAAAATAAAGTATTGGTA 3 9839 9858 2545 567254 N/A N/A AGATACTTTGAAGATATGAA 0 9854 9873 2546 567255 N/A N/A TGGGAAGATACTTTGAAGAT 0 9859 9878 2547 567256 N/A N/A CTAATAATGTGGGAAGATAC 0 9868 9887 2548 567257 N/A N/A CATTGCAGATAATAGCTAAT 0 9883 9902 2549 567258 N/A N/A AAGTTGTCATTGCAGATAAT 0 9890 9909 2550 567259 N/A N/A TTTTAAAAGTTGTCATTGCA 7 9896 9915 2551 567260 N/A N/A ATTCGGATTTTTAAAAGTTG 5 9904 9923 2552 567261 N/A N/A TTATTTGGGATTCGGATTTT 15 9913 9932 2553 567262 N/A N/A TTATAGTTAAGAGGTTTTCG 27 9949 9968 2554 567263 N/A N/A TTTCATTATAGTTAAGAGGT 12 9954 9973 2555 567264 N/A N/A GAACACTTTCATTATAGTTA 13 9960 9979 2556 567265 N/A N/A GAACTAGAATGAACACTTTC 28 9970 9989 2557 567266 N/A N/A TGATTGAACTAGAATGAACA 23 9975 9994 2558 567267 N/A N/A ATACCTGATTGAACTAGAAT 9 9980 9999 2559 567268 N/A N/A GTAAAATACCTGATTGAACT 6 9985 10004 2560 567269 N/A N/A TAGAGGTAAAATACCTGATT 16 9990 10009 2561 567270 N/A N/A AAGATTAGAGGTAAAATACC 0 9995 10014 2562 567271 N/A N/A TGAGGAAGATTAGAGGTAAA 6 10000 10019 2563 567272 N/A N/A GAAAATCTGAGGAAGATTAG 0 10007 10026 2564 567273 N/A N/A AAATAGAAAATCTGAGGAAG 0 10012 10031 2565 567274 N/A N/A ATCTATACACTACCAAAAAA 0 10029 10048 2566 567275 N/A N/A AAATAATCTATACACTACCA 19 10034 10053 2567 567276 N/A N/A AAATAATCTGTATAAATAAT 3 10047 10066 2568 567277 N/A N/A CCCAATTTTAAATAATCTGT 24 10056 10075 2569 567278 N/A N/A TAAGTCCCAATTTTAAATAA 0 10061 10080 2570 567279 N/A N/A TCTGTATAAGTCCCAATTTT 15 10067 10086 2571 567280 N/A N/A AATAATCTGTATAAGTCCCA 47 10072 10091 2572 567281 N/A N/A AGTTTTAAATAATCTGTATA 0 10079 10098 2573 567282 N/A N/A ATCCCAGTTTTAAATAATCT 6 10084 10103 2574 567283 N/A N/A CATGTATCCCAGTTTTAAAT 6 10089 10108 2575 567284 N/A N/A TAGATGCATGTATCCCAGTT 41 10095 10114 2576 567285 N/A N/A TGTTTTAGATGCATGTATCC 4 10100 10119 2577 567286 N/A N/A TACAGTGTTTTAGATGCATG 25 10105 10124 2578 567287 N/A N/A AATATTACAGTGTTTTAGAT 0 10110 10129 2579 567288 N/A N/A CTTATAAATATTACAGTGTT 2 10116 10135 2580 567289 N/A N/A CTTCCTTTCTTATAAATATT 12 10124 10143 2581 567290 N/A N/A TTTATCTTCCTTTCTTATAA 0 10129 10148 2582 567291 N/A N/A CGTAAGTTTATCTTCCTTTC 61 10135 10154 91 567292 N/A N/A TTCCCCGTAAGTTTATCTTC 22 10140 10159 2583 567293 N/A N/A TGTATTTCCCCGTAAGTTTA 0 10145 10164 2584 567294 N/A N/A GTTACTGTATTTCCCCGTAA 43 10150 10169 2585 544120 707 726 AGTTCTTGGTGCTCTTGGCT 80 6720 6739 15 337487 804 823 CACTTGTATGTTCACCTCTG 80 7389 7408 28

TABLE 137 Inhibition of ANGPTL3 mRNA by 5-10-5 MOE gapmers targeting SEQ ID NO: 1 and 2 SEQ SEQ SEQ ID ID SEQ ID ID NO: 1 NO: NO: 2 NO: 2 SEQ ISIS Start 1 Stop % Start Stop ID NO Site Site Sequence inhibition Site Site NO 563780 N/A N/A TGTTTTCTTCTGGAAGCAGA 10 3100 3119 2586 568085 N/A N/A CAGACCTAGACTTCTTAACT 8 3084 3103 2587 568086 N/A N/A AGCAGACCTAGACTTCTTAA 6 3086 3105 2588 568087 N/A N/A TTTTCTTCTGGAAGCAGACC 0 3098 3117 2589 568088 N/A N/A AAACATATATACATGCTTGT 52 11323 11342 2590 568089 N/A N/A TTAAACATATATACATGCTT 39 11325 11344 2591 568090 N/A N/A GTTTATTGAATTTTAAACAT 0 11337 11356 2592 568091 N/A N/A TTGTTTATTGAATTTTAAAC 9 11339 11358 2593 568092 N/A N/A CTTTGTTTATTGAATTTTAA 0 11341 11360 2594 568093 N/A N/A GTCTTTGTTTATTGAATTTT 28 11343 11362 2595 568094 N/A N/A GGGTCTTTGTTTATTGAATT 0 11345 11364 2596 568095 N/A N/A CTGGGTCTTTGTTTATTGAA 11 11347 11366 2597 568096 N/A N/A GACTGGGTCTTTGTTTATTG 35 11349 11368 2598 568097 N/A N/A TTTCTATAATTTAGGGACTG 12 11364 11383 2599 568098 N/A N/A AATTTCTATAATTTAGGGAC 0 11366 11385 2600 568099 N/A N/A TAAATTTCTATAATTTAGGG 5 11368 11387 2601 568100 N/A N/A CAAGAATAATTTAAATTTCT 38 11379 11398 2602 568101 N/A N/A GATAAACATGCAAGAATAAT 1 11389 11408 2603 568102 N/A N/A TCGATAAACATGCAAGAATA 51 11391 11410 2604 568103 N/A N/A TGTCGATAAACATGCAAGAA 37 11393 11412 2605 568104 N/A N/A GATGTCGATAAACATGCAAG 57 11395 11414 2606 568105 N/A N/A GTGATGTCGATAAACATGCA 61 11397 11416 2607 568106 N/A N/A TTGTGATGTCGATAAACATG 57 11399 11418 2608 568107 N/A N/A TGTTGTGATGTCGATAAACA 47 11401 11420 2609 568108 N/A N/A TCTGTTGTGATGTCGATAAA 53 11403 11422 2610 568109 N/A N/A GATCTGTTGTGATGTCGATA 36 11405 11424 2611 568110 N/A N/A GGGATCTGTTGTGATGTCGA 41 11407 11426 2612 568111 N/A N/A TAGGGATCTGTTGTGATGTC 43 11409 11428 2613 568112 N/A N/A TTTAGGGATCTGTTGTGATG 18 11411 11430 2614 568113 N/A N/A GATTTAGGGATCTGTTGTGA 41 11413 11432 2615 568114 N/A N/A ATCTAATCTTTAGGGATTTA 37 11435 11454 2616 568115 N/A N/A TTTGTATCTAATCTTTAGGG 28 11440 11459 2617 568116 N/A N/A AATTTGTATCTAATCTTTAG 0 11442 11461 2618 568117 N/A N/A GTGGTAAAAAATTTGTATCT 13 11451 11470 2619 568118 N/A N/A CTGTGGTAAAAAATTTGTAT 5 11453 11472 2620 568119 N/A N/A TACTGTGGTAAAAAATTTGT 10 11455 11474 2621 568120 N/A N/A GATACTGTGGTAAAAAATTT 17 11457 11476 2622 568121 N/A N/A AGTGATACTGTGGTAAAAAA 38 11460 11479 2623 568122 N/A N/A CAAGTGATACTGTGGTAAAA 58 11462 11481 2624 568123 N/A N/A GACAAGTGATACTGTGGTAA 52 11464 11483 2625 568124 N/A N/A CTGACAAGTGATACTGTGGT 62 11466 11485 2626 568125 N/A N/A TTCTGACAAGTGATACTGTG 27 11468 11487 2627 568126 N/A N/A AATTCTGACAAGTGATACTG 33 11470 11489 2628 568127 N/A N/A ATAAATTCTGACAAGTGATA 38 11473 11492 2629 568128 N/A N/A CTGGCAGTTTTAAAAAATCA 28 11502 11521 2630 568129 N/A N/A TTCTTACTGGCAGTTTTAAA 56 11508 11527 2631 568130 N/A N/A ATTTCTTACTGGCAGTTTTA 47 11510 11529 2632 568131 N/A N/A AAATTTCTTACTGGCAGTTT 53 11512 11531 2633 568132 N/A N/A TTTAAAATTTCTTACTGGCA 46 11516 11535 2634 568133 N/A N/A TTAATTTAAAATTTCTTACT 9 11520 11539 2635 568134 N/A N/A CAAATGGGTTTAATTTAAAA 1 11529 11548 2636 568135 N/A N/A AACAAATGGGTTTAATTTAA 11 11531 11550 2637 568136 N/A N/A TTAACAAATGGGTTTAATTT 12 11533 11552 2638 568137 N/A N/A CTTTAACAAATGGGTTTAAT 27 11535 11554 2639 568138 N/A N/A TCCTTTAACAAATGGGTTTA 52 11537 11556 2640 568139 N/A N/A CTATATCCTTTAACAAATGG 24 11542 11561 2641 568140 N/A N/A GGGCACTATATCCTTTAACA 45 11547 11566 2642 568141 N/A N/A TTGGGCACTATATCCTTTAA 20 11549 11568 2643 568142 N/A N/A TATAACTTGGGCACTATATC 27 11555 11574 2644 568143 N/A N/A CATATAACTTGGGCACTATA 40 11557 11576 2645 568144 N/A N/A ACCATATAACTTGGGCACTA 69 11559 11578 103 568145 N/A N/A TCACCATATAACTTGGGCAC 60 11561 11580 2646 568146 N/A N/A GGTCACCATATAACTTGGGC 73 11563 11582 104 568147 N/A N/A TAGGTCACCATATAACTTGG 51 11565 11584 2647 568148 N/A N/A GGTAGGTCACCATATAACTT 57 11567 11586 2648 568149 N/A N/A AAGGTAGGTCACCATATAAC 52 11569 11588 2649 568150 N/A N/A CAAAGGTAGGTCACCATATA 28 11571 11590 2650 568151 N/A N/A GACAAAGGTAGGTCACCATA 67 11573 11592 105 568152 N/A N/A GTATTGACAAAGGTAGGTCA 55 11578 11597 2651 568153 N/A N/A AAGTATTGACAAAGGTAGGT 36 11580 11599 2652 568154 N/A N/A CTAAGTATTGACAAAGGTAG 24 11582 11601 2653 568155 N/A N/A TGCTAAGTATTGACAAAGGT 49 11584 11603 2654 568156 N/A N/A AATGCTAAGTATTGACAAAG 10 11586 11605 2655 568157 N/A N/A CATAATGCTAAGTATTGACA 19 11589 11608 2656 568158 N/A N/A TACATAATGCTAAGTATTGA 4 11591 11610 2657 568159 N/A N/A AATACATAATGCTAAGTATT 1 11593 11612 2658 568160 N/A N/A GAAATACATAATGCTAAGTA 23 11595 11614 2659 568161 N/A N/A TTTGAAATACATAATGCTAA 8 11598 11617 2660 568162 N/A N/A GGATAATTTGAAATACATAA 16 11604 11623 2661 568163 N/A N/A TTGGATAATTTGAAATACAT 0 11606 11625 2662 568164 N/A N/A TATTGGATAATTTGAAATAC 0 11608 11627 2663 568165 N/A N/A ATCCAGTTAAAGCTTGTAAA 46 4466 4485 2664 568166 N/A N/A TCATGATCCAGTTAAAGCTT 32 4471 4490 2665 568167 N/A N/A TTTACTCATGATCCAGTTAA 24 4476 4495 2666 568168 N/A N/A GATAATTTTACTCATGATCC 53 4482 4501 2667 568169 N/A N/A GATGTGATAATTTTACTCAT 27 4487 4506 2668 568170 N/A N/A ATGCTGATGTGATAATTTTA 42 4492 4511 2669 568171 N/A N/A CAGTTATGCTGATGTGATAA 0 4497 4516 2670 568172 N/A N/A TTTAACAGTTATGCTGATGT 17 4502 4521 2671 568173 N/A N/A GCAATTTTAACAGTTATGCT 11 4507 4526 2672 568174 N/A N/A AGAGCCTGCAATTTTAACAG 25 4514 4533 2673 568175 N/A N/A GCTTCAGAGCCTGCAATTTT 47 4519 4538 2674 568176 N/A N/A TATTAGCTTCAGAGCCTGCA 48 4524 4543 2675 568177 N/A N/A TAGTTTATTAGCTTCAGAGC 20 4529 4548 2676 568178 N/A N/A GCAGGTAGTTTATTAGCTTC 39 4534 4553 2677 568179 N/A N/A TAAATGCAGGTAGTTTATTA 0 4539 4558 2678 568180 N/A N/A ATGGTTTAAATGCAGGTAGT 20 4545 4564 2679 568181 N/A N/A GAGCCATGGTTTAAATGCAG 33 4550 4569 2680 568182 N/A N/A TTTTAGAGCCATGGTTTAAA 40 4555 4574 2681 568183 N/A N/A CAAAGTTTTAGAGCCATGGT 54 4560 4579 2682 568184 N/A N/A TCACACAAAGTTTTAGAGCC 61 4565 4584 2683 568185 N/A N/A CAAGGTCACACAAAGTTTTA 17 4570 4589 2684 568186 N/A N/A GGGTGAAGTAATTTATTCAA 0 4587 4606 2685 568187 N/A N/A GTGAGGAAACTGAGAGATAA 12 4609 4628 2686 568188 N/A N/A TGTAGTATATGTGAGGAAAC 38 4619 4638 2687 568189 N/A N/A ATCTTTGTAGTATATGTGAG 30 4624 4643 2688 568190 N/A N/A TTATTATCTTTGTAGTATAT 19 4629 4648 2689 568191 N/A N/A TTCTGTTATTATCTTTGTAG 48 4634 4653 2690 568192 N/A N/A ATAAGTTCTGTTATTATCTT 16 4639 4658 2691 568193 N/A N/A ATCCTATAAGTTCTGTTATT 22 4644 4663 2692 568194 N/A N/A CAATAATCCTATAAGTTCTG 0 4649 4668 2693 568195 N/A N/A TAAGATGACATTGGCTGCTA 49 4689 4708 2694 568196 N/A N/A TTTAGTAAGATGACATTGGC 32 4694 4713 2695 568197 N/A N/A TTGAATTTTAGTAAGATGAC 19 4700 4719 2696 568198 N/A N/A CTAATTTGAATTTTAGTAAG 34 4705 4724 2697 568199 N/A N/A CATGATCTAATTTGAATTTT 29 4711 4730 2698 568200 N/A N/A CAAAGAGAAACATGATCTAA 27 4721 4740 2699 568201 N/A N/A GTTTTGAGCAAAGAGAAACA 36 4729 4748 2700 568202 N/A N/A GTGTGGTTTTGAGCAAAGAG 3 4734 4753 2701 568203 N/A N/A AGCTATTGTGTGGTTTTGAG 13 4741 4760 2702 568204 N/A N/A TGAAATGGAAAGCTATTGTG 15 4751 4770 2703 568205 N/A N/A TATGAGTGAAATGGAAAGCT 27 4757 4776 2704 568206 N/A N/A GCCAATATGAGTGAAATGGA 62 4762 4781 106 568207 N/A N/A AAAGAGCCAATATGAGTGAA 25 4767 4786 2705 568208 N/A N/A TTGGTCTAAAGAGCCAATAT 42 4774 4793 2706 568209 N/A N/A GGTAATCTTGGTCTAAAGAG 29 4781 4800 2707 568210 N/A N/A GTGAGATGACGAAGGGTTGG 0 4800 4819 2708 568211 N/A N/A AGTCAGTGAGATGACGAAGG 5 4805 4824 2709 568212 N/A N/A GGTGAAGTCAGTGAGATGAC 12 4810 4829 2710 568213 N/A N/A GTAGAGGAGGTGAAGTCAGT 13 4818 4837 2711 568214 N/A N/A AACTAGAGTAGAGGAGGTGA 20 4825 4844 2712 568215 N/A N/A AGAATAACTAGAGTAGAGGA 33 4830 4849 2713 568216 N/A N/A CGGTCAGAATAACTAGAGTA 39 4835 4854 2714 568217 N/A N/A TAAAGCGGTCAGAATAACTA 29 4840 4859 2715 568218 N/A N/A ACTGGTAAAGCGGTCAGAAT 17 4845 4864 2716 568219 N/A N/A TGAATACTGGTAAAGCGGTC 37 4850 4869 2717 568220 N/A N/A TGTGTTTGAATACTGGTAAA 21 4856 4875 2718 568221 N/A N/A AGTATGTTTGATGTGTTTGA 25 4867 4886 2719 568222 N/A N/A GTGGCAGTATGTTTGATGTG 15 4872 4891 2720 568223 N/A N/A TTGAGGTGGCAGTATGTTTG 14 4877 4896 2721 568224 N/A N/A AGGCTTTGAGGTGGCAGTAT 33 4882 4901 2722 568225 N/A N/A GGCAAAGGCTTTGAGGTGGC 27 4887 4906 2723 568226 N/A N/A AACAAGGGCAAAGGCTTTGA 24 4893 4912 2724 568227 N/A N/A TAGAGGAAACAACAAGGGCA 24 4903 4922 2725 568228 N/A N/A CCAGTTAGAGGAAACAACAA 4 4908 4927 2726 568229 N/A N/A GATACCAGGGCAGAAGAGCG 24 4930 4949 2727 568230 N/A N/A AAATCAGAGAGTGGGCCACG 24 4952 4971 2728 568231 N/A N/A CCTAAGGGAAATCAGAGAGT 19 4960 4979 2729 568232 N/A N/A ACGACCCTAAGGGAAATCAG 30 4965 4984 2730 568233 N/A N/A TGATAACGACCCTAAGGGAA 0 4970 4989 2731 568234 N/A N/A TTTTGTTTGATAACGACCCT 22 4977 4996 2732 568235 N/A N/A GTCTTCATTGGGAATTTTTT 37 4993 5012 2733 568236 N/A N/A TGTAAGTCTTCATTGGGAAT 23 4998 5017 2734 568237 N/A N/A GACCTTGTAAGTCTTCATTG 52 5003 5022 2735 568238 N/A N/A TAAGTGACCTTGTAAGTCTT 36 5008 5027 2736 568239 N/A N/A TTGGTTAAGTGACCTTGTAA 11 5013 5032 2737 568240 N/A N/A TGATTTTTGGTTAAGTGACC 12 5019 5038 2738 568241 N/A N/A GGTTGTGATTTTTGGTTAAG 11 5024 5043 2739 568242 N/A N/A CAGGCGGTTGTGATTTTTGG 41 5029 5048 2740 568243 N/A N/A GGGACCAGGCGGTTGTGATT 22 5034 5053 2741 568244 N/A N/A CTAAGGAAGTAGAAGTTTTC 42 5060 5079 2742 568245 N/A N/A AGTAGCTAAGGAAGTAGAAG 11 5065 5084 2743 568246 N/A N/A CAGGAGAAAAGTAGCTAAGG 36 5074 5093 2744 568247 N/A N/A GTGTGCAGGAGAAAAGTAGC 14 5079 5098 2745 568248 N/A N/A TAAAGGTGAGTGTGCAGGAG 7 5088 5107 2746 568249 N/A N/A ATGTTAAATAAAGGTGAGTG 8 5096 5115 2747 568250 N/A N/A ATGTTATGTTAAATAAAGGT 27 5101 5120 2748 568251 N/A N/A AATTTATGTTATGTTAAATA 27 5106 5125 2749 568252 N/A N/A TAACTAAAATTTATGTTATG 28 5113 5132 2750 568253 N/A N/A GATAAATAACTAAAATTTAT 32 5119 5138 2751 568254 N/A N/A TTTAGTGCAGGAATAGAAGA 33 5139 5158 2752 568255 N/A N/A AATCCCTGTATTCACAGAGC 68 5165 5184 2753 568256 N/A N/A GAAAAAATCCCTGTATTCAC 0 5170 5189 2754 568257 N/A N/A TAATGGAAAAAATCCCTGTA 8 5175 5194 2755 568258 N/A N/A AAATATGAAGATAATGGAAA 26 5186 5205 2756 568259 N/A N/A ATAATGGAAAATATGAAGAT 18 5194 5213 2757 568260 N/A N/A TATACAAATAATGGAAAATA 30 5201 5220 2758 568261 N/A N/A TTCTGGAGTATATACAAATA 45 5211 5230 2759 568262 N/A N/A ATTCTATATTCTGGAGTATA 40 5219 5238 2760 568263 N/A N/A CCATACAGTATTCTATATTC 57 5228 5247 2761 568264 N/A N/A CTGTGTGCCATACAGTATTC 28 5235 5254 2762 568265 N/A N/A GCCTACTGTGTGCCATACAG 60 5240 5259 2763 568266 N/A N/A AGAAATGCCTACTGTGTGCC 42 5246 5265 2764 568267 N/A N/A TCAACAGAAATGCCTACTGT 52 5251 5270 2765 568268 N/A N/A ATTAATTCAACAGAAATGCC 46 5257 5276 2766 568269 N/A N/A GACATTACATTTATTAATTC 32 5269 5288 2767 568270 N/A N/A GTGAATATGACATTACATTT 32 5277 5296 2768 568271 N/A N/A CTTCTGTGTGAATATGACAT 50 5284 5303 2769 568272 N/A N/A ACACGCTTCTGTGTGAATAT 43 5289 5308 2770 568273 N/A N/A ATAGCACACGCTTCTGTGTG 31 5294 5313 2771 568274 N/A N/A TAATCATAGCACACGCTTCT 40 5299 5318 2772 568275 N/A N/A AATAATAATCATAGCACACG 20 5304 5323 2773 568276 N/A N/A CCAAGTAATAATAATCATAG 35 5310 5329 2774 568277 N/A N/A CTAGTAATCCAAGTAATAAT 38 5318 5337 2775 568278 N/A N/A TATTTCTAGTAATCCAAGTA 39 5323 5342 2776 568279 N/A N/A CACACTATTTCTAGTAATCC 51 5328 5347 2777 568280 N/A N/A TTATGAGGCACACTATTTCT 25 5336 5355 2778 568281 N/A N/A TTTAATTATGAGGCACACTA 35 5341 5360 2779 568282 N/A N/A GTTGACCTTTAATTATGAGG 63 5348 5367 2780 568283 N/A N/A TTACATTGTTGAATGTTGAC 45 5362 5381 2781 568284 N/A N/A ATTAATTACATTGTTGAATG 31 5367 5386 2782 568285 N/A N/A TGTAGATTAATTACATTGTT 49 5372 5391 2783 568286 N/A N/A TACATTGTAGATTAATTACA 43 5377 5396 2784 568287 N/A N/A AGATGTTTACATTGTAGATT 28 5384 5403 2785 568288 N/A N/A TTCACCAGATGTTTACATTG 36 5390 5409 2786 568289 N/A N/A GTCACTTCACCAGATGTTTA 65 5395 5414 2787 568290 N/A N/A CCTCTGTCACTTCACCAGAT 67 5400 5419 2788 568291 N/A N/A GCTTCCCTCTGTCACTTCAC 70 5405 5424 2789 568292 N/A N/A CAAGTGCTTCCCTCTGTCAC 33 5410 5429 2790 568293 N/A N/A TTTCTAAACAAGTGCTTCCC 70 5418 5437 107 568294 N/A N/A GCTTTTTTCTAAACAAGTGC 45 5423 5442 2791 568295 N/A N/A ACATAGCTTTTTTCTAAACA 9 5428 5447 2792 568296 N/A N/A TTCTGACATAGCTTTTTTCT 23 5433 5452 2793 568297 N/A N/A ATGGATTCTGACATAGCTTT 46 5438 5457 2794 568298 N/A N/A AATACATGGATTCTGACATA 37 5443 5462 2795 568299 N/A N/A ATTAGAATACATGGATTCTG 57 5448 5467 2796 568300 N/A N/A CTGCATATTAGAATACATGG 75 5454 5473 108 568301 N/A N/A TTGTACTGCATATTAGAATA 53 5459 5478 2797 568302 N/A N/A AACTATTGTACTGCATATTA 25 5464 5483 2798 568303 N/A N/A TTTTAAACTATTGTACTGCA 25 5469 5488 2799 568304 N/A N/A TGAGAGTATTATTAATATTT 8 5487 5506 2800 568305 N/A N/A GCTGTTTGAGAGTATTATTA 50 5493 5512 2801 568306 N/A N/A GAATAGCTGTTTGAGAGTAT 38 5498 5517 2802 568307 N/A N/A CCTCTTGAATAGCTGTTTGA 55 5504 5523 2803 568308 N/A N/A TGAATCCTCTTGAATAGCTG 55 5509 5528 2804 568309 N/A N/A TTTTTTGAATCCTCTTGAAT 46 5514 5533 2805 568310 N/A N/A TTATGTTTTTTGAATCCTCT 36 5519 5538 2806 568311 N/A N/A GTTTATATTATGTTTTTTGA 6 5526 5545 2807 568312 N/A N/A TCTGAGTTTATATTATGTTT 29 5531 5550 2808 568313 N/A N/A CAGTTTCTCTGAGTTTATAT 28 5538 5557 2809 568314 N/A N/A GTTTACCAGTTTCTCTGAGT 44 5544 5563 2810 568315 N/A N/A ATTTTGTTTACCAGTTTCTC 58 5549 5568 2811 568316 N/A N/A AAATGATTTTGTTTACCAGT 29 5554 5573 2812 568317 N/A N/A CTCTTGAAAATGATTTTGTT 22 5561 5580 2813 568318 N/A N/A TATATCTCTTGAAAATGATT 5 5566 5585 2814 568319 N/A N/A CAGGTTGGCAAGTTTGTTTG 27 6175 6194 2815 568320 N/A N/A GTTGGCAGGTTGGCAAGTTT 44 6180 6199 2816 568321 N/A N/A ATATCTGTAGATGTTGGCAG 59 6192 6211 2817 568322 N/A N/A TAAACATATCTGTAGATGTT 18 6197 6216 2818 568323 N/A N/A ACCTGTAAACATATCTGTAG 57 6202 6221 2819 568324 N/A N/A TTTTGACCTGTAAACATATC 23 6207 6226 2820 568325 N/A N/A ATAATTTTTGACCTGTAAAC 7 6212 6231 2821 568326 N/A N/A TAATTTGATAATTTTTGACC 7 6219 6238 2822 568327 N/A N/A TTCTTGATAATTTGATAATT 8 6226 6245 2823 568328 N/A N/A ACCAGGCTTTCTTGATAATT 55 6234 6253 2824 568329 N/A N/A TTTGAACCAGGCTTTCTTGA 49 6239 6258 2825 568330 N/A N/A CATAATTTGAACCAGGCTTT 68 6244 6263 109 568331 N/A N/A AGACATAATACATAATTTGA 8 6254 6273 2826 568332 N/A N/A CTGTGATAAAGACATAATAC 40 6263 6282 2827 568333 N/A N/A CAGACCTGTGATAAAGACAT 16 6268 6287 2828 568334 N/A N/A ATCTTCAGACCTGTGATAAA 7 6273 6292 2829 568335 N/A N/A TACTGATCTTCAGACCTGTG 47 6278 6297 2830 568336 N/A N/A TTAATAATTTTCAGTTTTAG 35 6302 6321 2831 568337 N/A N/A TAAGTTTAATAATTTTCAGT 23 6307 6326 2832 568338 N/A N/A TTCAGATTTTAAGTTTAATA 10 6316 6335 2833 568339 N/A N/A TATATTTGATATTCTGTTCA 42 6332 6351 2834 568340 N/A N/A ATATTGTAATGTATTCTTTT 0 6368 6387 2835 568341 N/A N/A TTAGAATATTGTAATGTATT 19 6373 6392 2836 568342 N/A N/A TTTGCTTAGAATATTGTAAT 9 6378 6397 2837 568343 N/A N/A ACTGCTTTGCTTAGAATATT 36 6383 6402 2838 568344 N/A N/A AAGTAGAGACTGCTTTGCTT 60 6391 6410 2839 568345 N/A N/A GCAAGGCCAAAAGTAGAGAC 59 6401 6420 2840 568346 N/A N/A ACAGAGCAAGGCCAAAAGTA 45 6406 6425 2841 568347 N/A N/A GGAAAACAGAGCAAGGCCAA 49 6411 6430 2842 568348 N/A N/A TGGTCGGAAAACAGAGCAAG 38 6416 6435 2843 568349 N/A N/A GACATTGGTCGGAAAACAGA 26 6421 6440 2844 568350 N/A N/A AAGCAGACATTGGTCGGAAA 50 6426 6445 2845 568351 N/A N/A CAAGGCAAAAAAGCAGACAT 39 6436 6455 2846 568352 N/A N/A ATAAAGCAAGGCAAAAAAGC 20 6442 6461 2847 568353 N/A N/A CATTATTTAATAAGATAAAA 29 6464 6483 2848 568354 N/A N/A AAATATTTAATCAGGGACAT 35 6481 6500 2849 568355 N/A N/A TGTTCTCAAAATATTTAATC 32 6489 6508 2850 568356 N/A N/A GATTACCTGTTCTCAAAATA 40 6496 6515 2851 568357 N/A N/A GATTGTACAGATTACCTGTT 12 6505 6524 2852 568358 N/A N/A ATTCAGATTGTACAGATTAC 34 6510 6529 2853 568359 N/A N/A AAACAGTGTTATTCAGATTG 32 6520 6539 2854 568360 N/A N/A TAGATAAACAGTGTTATTCA 25 6525 6544 2855 568361 N/A N/A ATATTTAGATAAACAGTGTT 14 6530 6549 2856 568362 N/A N/A GTTTGATATTTAGATAAACA 27 6535 6554 2857 568363 N/A N/A AACGGTGTTTGATATTTAGA 33 6541 6560 2858 568364 N/A N/A GTTATAACGGTGTTTGATAT 29 6546 6565 2859 568365 N/A N/A ATAATGTTATAACGGTGTTT 21 6551 6570 2860 568366 N/A N/A AGTTCATAATGTTATAACGG 37 6556 6575 2861 568367 N/A N/A CTTTCAGTTCATAATGTTAT 46 6561 6580 2862 568368 N/A N/A AGTACAGTTTGTCTTTCAGT 48 6573 6592 2863 568369 N/A N/A TCAGAAGTACAGTTTGTCTT 47 6578 6597 2864 568370 N/A N/A GGATGTCAGAAGTACAGTTT 46 6583 6602 2865 568371 N/A N/A GAGTAAGGATGTCAGAAGTA 45 6589 6608 2866 568372 N/A N/A GAAATCTGAGTAAGGATGTC 31 6596 6615 2867 568373 N/A N/A TACTGAATATACAATTAGGG 5 6616 6635 2868 568374 N/A N/A AATGATACTGAATATACAAT 21 6621 6640 2869 568375 N/A N/A GAATATAAATCTGTTTTTTA 19 6642 6661 2870 568376 N/A N/A TAAAAGAATATAAATCTGTT 32 6647 6666 2871 568377 N/A N/A GCTGATAAAAGAATATAAAT 50 6652 6671 2872 568378 N/A N/A CCTTCTGAGCTGATAAAAGA 37 6660 6679 2873 568379 N/A N/A CTAGTCCTTCTGAGCTGATA 45 6665 6684 2874 568380 N/A N/A TTACCATCATGTTTTACATT 30 6770 6789 2875 568381 N/A N/A CAAAGTGTCTTACCATCATG 24 6779 6798 2876 568382 N/A N/A AAACCCACCAAAGTGTCTTA 15 6787 6806 2877 568383 N/A N/A AGAAGGAAACCCACCAAAGT 22 6793 6812 2878 568384 N/A N/A AATAATAGCTTCAAGAAGGA 25 6806 6825 2879 568385 N/A N/A AATTTGATAATAATAGCTTC 24 6814 6833 2880 568386 N/A N/A TAGGGAATTTGATAATAATA 20 6819 6838 2881 568387 N/A N/A AAGAATAGGGAATTTGATAA 0 6824 6843 2882 568388 N/A N/A GTCCTAAGAATAGGGAATTT 45 6829 6848 2883 568389 N/A N/A TAGAACAAGTCCTAAGAATA 21 6837 6856 2884 568390 N/A N/A TTAGTCTAGAACAAGTCCTA 28 6843 6862 2885 568391 N/A N/A ATCTTTTAGTCTAGAACAAG 21 6848 6867 2886 568392 N/A N/A TAACTATCTTTTAGTCTAGA 13 6853 6872 2887 568393 N/A N/A ATCTCTTAACTATCTTTTAG 28 6859 6878 2888 568394 N/A N/A TGGATATCTCTTAACTATCT 48 6864 6883 2889 568395 N/A N/A TTTGATGGATATCTCTTAAC 35 6869 6888 2890 544120 707 726 AGTTCTTGGTGCTCTTGGCT 80 6720 6739 15 337487 804 823 CACTTGTATGTTCACCTCTG 76 7389 7408 28 568006 2014 2033 TTAATTCTGCTTCATTAGGT 53 10986 11005 2891 568007 2015 2034 TTTAATTCTGCTTCATTAGG 38 10987 11006 2892 568008 2020 2039 CAGTATTTAATTCTGCTTCA 56 10992 11011 2893 568009 2021 2040 ACAGTATTTAATTCTGCTTC 63 10993 11012 2894 568010 2022 2041 TACAGTATTTAATTCTGCTT 56 10994 11013 2895 568011 2023 2042 ATACAGTATTTAATTCTGCT 39 10995 11014 2896 568012 2024 2043 AATACAGTATTTAATTCTGC 21 10996 11015 2897 568013 2025 2044 TAATACAGTATTTAATTCTG 12 10997 11016 2898 568014 2027 2046 TTTAATACAGTATTTAATTC 0 10999 11018 2899 568015 2028 2047 TTTTAATACAGTATTTAATT 15 11000 11019 2900 568016 2031 2050 TTATTTTAATACAGTATTTA 0 11003 11022 2901 568017 2034 2053 AACTTATTTTAATACAGTAT 24 11006 11025 2902 568018 2035 2054 GAACTTATTTTAATACAGTA 21 11007 11026 2903 568019 2036 2055 CGAACTTATTTTAATACAGT 2 11008 11027 2904 568020 2037 2056 GCGAACTTATTTTAATACAG 54 11009 11028 2905 568021 2038 2057 AGCGAACTTATTTTAATACA 35 11010 11029 2906 568022 2039 2058 CAGCGAACTTATTTTAATAC 50 11011 11030 2907 568023 2040 2059 ACAGCGAACTTATTTTAATA 34 11012 11031 2908 568024 2041 2060 GACAGCGAACTTATTTTAAT 52 11013 11032 2909 568025 2042 2061 AGACAGCGAACTTATTTTAA 58 11014 11033 2910 568026 2044 2063 AAAGACAGCGAACTTATTTT 32 11016 11035 2911 568027 2045 2064 TAAAGACAGCGAACTTATTT 26 11017 11036 2912 568028 2048 2067 GTTTAAAGACAGCGAACTTA 62 11020 11039 2913 568029 2049 2068 TGTTTAAAGACAGCGAACTT 58 11021 11040 2914 568030 2050 2069 TTGTTTAAAGACAGCGAACT 52 11022 11041 2915 568031 2051 2070 TTTGTTTAAAGACAGCGAAC 61 11023 11042 2916 568032 2052 2071 ATTTGTTTAAAGACAGCGAA 41 11024 11043 2917 568033 2053 2072 CATTTGTTTAAAGACAGCGA 60 11025 11044 2918 568034 2054 2073 CCATTTGTTTAAAGACAGCG 88 11026 11045 98 568035 2055 2074 TCCATTTGTTTAAAGACAGC 57 11027 11046 2919 568036 2056 2075 CTCCATTTGTTTAAAGACAG 58 11028 11047 2920 568037 2058 2077 ATCTCCATTTGTTTAAAGAC 56 11030 11049 2921 568038 2059 2078 CATCTCCATTTGTTTAAAGA 54 11031 11050 2922 568039 2060 2079 TCATCTCCATTTGTTTAAAG 62 11032 11051 2923 568040 2061 2080 GTCATCTCCATTTGTTTAAA 53 11033 11052 2924 568041 2063 2082 TAGTCATCTCCATTTGTTTA 48 11035 11054 2925 568042 2064 2083 GTAGTCATCTCCATTTGTTT 44 11036 11055 2926 568043 2065 2084 AGTAGTCATCTCCATTTGTT 48 11037 11056 2927 568044 2066 2085 TAGTAGTCATCTCCATTTGT 45 11038 11057 2928 568045 2067 2086 TTAGTAGTCATCTCCATTTG 66 11039 11058 2929 568046 2068 2087 CTTAGTAGTCATCTCCATTT 66 11040 11059 2930 568047 2069 2088 ACTTAGTAGTCATCTCCATT 68 11041 11060 99 568048 2070 2089 GACTTAGTAGTCATCTCCAT 77 11042 11061 100 568049 2071 2090 TGACTTAGTAGTCATCTCCA 70 11043 11062 101 568050 2072 2091 GTGACTTAGTAGTCATCTCC 65 11044 11063 2931 568051 2073 2092 TGTGACTTAGTAGTCATCTC 49 11045 11064 2932 568052 2074 2093 ATGTGACTTAGTAGTCATCT 47 11046 11065 2933 568053 2075 2094 AATGTGACTTAGTAGTCATC 48 11047 11066 2934 568054 2076 2095 CAATGTGACTTAGTAGTCAT 60 11048 11067 2935 568055 2077 2096 TCAATGTGACTTAGTAGTCA 54 11049 11068 2936 568056 2078 2097 GTCAATGTGACTTAGTAGTC 72 11050 11069 102 568057 2079 2098 AGTCAATGTGACTTAGTAGT 62 11051 11070 2937 568058 2083 2102 TTAAAGTCAATGTGACTTAG 15 11055 11074 2938 568059 2084 2103 GTTAAAGTCAATGTGACTTA 28 11056 11075 2939 568060 2085 2104 TGTTAAAGTCAATGTGACTT 35 11057 11076 2940 568061 2086 2105 ATGTTAAAGTCAATGTGACT 17 11058 11077 2941 568062 2087 2106 CATGTTAAAGTCAATGTGAC 27 11059 11078 2942 568063 2089 2108 CTCATGTTAAAGTCAATGTG 28 11061 11080 2943 568064 2090 2109 CCTCATGTTAAAGTCAATGT 50 11062 11081 2944 568066 2091 2110 ACCTCATGTTAAAGTCAATG 48 11063 11082 2945 568068 2092 2111 TACCTCATGTTAAAGTCAAT 13 11064 11083 2946 568069 2093 2112 ATACCTCATGTTAAAGTCAA 43 11065 11084 2947 568072 2094 2113 GATACCTCATGTTAAAGTCA 40 11066 11085 2948 568073 2095 2114 TGATACCTCATGTTAAAGTC 40 11067 11086 2949 568075 2096 2115 GTGATACCTCATGTTAAAGT 37 11068 11087 2950 568077 2097 2116 AGTGATACCTCATGTTAAAG 6 11069 11088 2951 568078 2098 2117 TAGTGATACCTCATGTTAAA 12 11070 11089 2952 568079 2099 2118 ATAGTGATACCTCATGTTAA 8 11071 11090 2953 568080 2100 2119 TATAGTGATACCTCATGTTA 13 11072 11091 2954 568081 2101 2120 GTATAGTGATACCTCATGTT 41 11073 11092 2955 568082 2102 2121 GGTATAGTGATACCTCATGT 53 11074 11093 2956 568083 2106 2125 ATAAGGTATAGTGATACCTC 54 11078 11097 2957 568084 2107 2126 AATAAGGTATAGTGATACCT 38 11079 11098 2958

TABLE 138 Inhibition of ANGPTL3 mRNA by 5-10-5 MOE gapmers targeting SEQ ID NO: 1 and 2 SEQ SEQ SEQ ID SEQ ID ID NO: NO: ID NO: NO: 2 SEQ ISIS 1 Start 1 Stop % 2 Start Stop ID NO Site Site Sequence inhibition Site Site NO 544120 707 726 AGTTCTTGGTGCTCTTGGCT 83 6720 6739 15 337487 804 823 CACTTGTATGTTCACCTCTG 81 7389 7408 28 567295 1452 1471 TAATGTTTAAATTATTGCCT 43 10424 10443 2959 567296 1455 1474 GGTTAATGTTTAAATTATTG 22 10427 10446 2960 567297 1456 1475 AGGTTAATGTTTAAATTATT 0 10428 10447 2961 567298 1457 1476 GAGGTTAATGTTTAAATTAT 0 10429 10448 2962 567299 1458 1477 TGAGGTTAATGTTTAAATTA 6 10430 10449 2963 567300 1460 1479 AATGAGGTTAATGTTTAAAT 14 10432 10451 2964 567301 1461 1480 GAATGAGGTTAATGTTTAAA 5 10433 10452 2965 567302 1462 1481 GGAATGAGGTTAATGTTTAA 27 10434 10453 2966 567303 1463 1482 TGGAATGAGGTTAATGTTTA 32 10435 10454 2967 567304 1464 1483 TTGGAATGAGGTTAATGTTT 37 10436 10455 2968 567305 1465 1484 CTTGGAATGAGGTTAATGTT 25 10437 10456 2969 567306 1468 1487 TAACTTGGAATGAGGTTAAT 29 10440 10459 2970 567307 1469 1488 TTAACTTGGAATGAGGTTAA 44 10441 10460 2971 337513 1470 1489 ATTAACTTGGAATGAGGTTA 52 10442 10461 2972 567308 1471 1490 CATTAACTTGGAATGAGGTT 62 10443 10462 2973 567309 1472 1491 ACATTAACTTGGAATGAGGT 58 10444 10463 2974 567310 1473 1492 CACATTAACTTGGAATGAGG 78 10445 10464 92 567311 1475 1494 ACCACATTAACTTGGAATGA 59 10447 10466 2975 567312 1476 1495 GACCACATTAACTTGGAATG 57 10448 10467 2976 337514 1477 1496 AGACCACATTAACTTGGAAT 71 10449 10468 2977 567313 1478 1497 TAGACCACATTAACTTGGAA 43 10450 10469 2978 567314 1479 1498 TTAGACCACATTAACTTGGA 59 10451 10470 2979 567315 1480 1499 ATTAGACCACATTAACTTGG 70 10452 10471 2980 567316 1481 1500 TATTAGACCACATTAACTTG 53 10453 10472 2981 567317 1482 1501 TTATTAGACCACATTAACTT 49 10454 10473 2982 567318 1483 1502 ATTATTAGACCACATTAACT 41 10455 10474 2983 567319 1484 1503 GATTATTAGACCACATTAAC 47 10456 10475 2984 567320 1487 1506 CCAGATTATTAGACCACATT 86 10459 10478 93 567321 1489 1508 TACCAGATTATTAGACCACA 85 10461 10480 94 337516 1490 1509 ATACCAGATTATTAGACCAC 77 10462 10481 86 567322 1491 1510 AATACCAGATTATTAGACCA 50 10463 10482 2985 567323 1492 1511 TAATACCAGATTATTAGACC 56 10464 10483 2986 567324 1494 1513 TTTAATACCAGATTATTAGA 9 10466 10485 2987 567325 1495 1514 ATTTAATACCAGATTATTAG 24 10467 10486 2988 567326 1496 1515 GATTTAATACCAGATTATTA 37 10468 10487 2989 567327 1500 1519 TAAGGATTTAATACCAGATT 60 10472 10491 2990 567328 1507 1526 TTTCTCTTAAGGATTTAATA 34 10479 10498 2991 567329 1508 1527 CTTTCTCTTAAGGATTTAAT 46 10480 10499 2992 567330 1509 1528 GCTTTCTCTTAAGGATTTAA 75 10481 10500 95 567331 1510 1529 AGCTTTCTCTTAAGGATTTA 59 10482 10501 2993 567332 1511 1530 AAGCTTTCTCTTAAGGATTT 30 10483 10502 2994 567333 1513 1532 TCAAGCTTTCTCTTAAGGAT 65 10485 10504 2995 567334 1514 1533 CTCAAGCTTTCTCTTAAGGA 77 10486 10505 96 567335 1515 1534 TCTCAAGCTTTCTCTTAAGG 75 10487 10506 97 567336 1516 1535 TTCTCAAGCTTTCTCTTAAG 59 10488 10507 2996 567337 1517 1536 TTTCTCAAGCTTTCTCTTAA 52 10489 10508 2997 567338 1521 1540 TCTATTTCTCAAGCTTTCTC 68 10493 10512 2998 567339 1522 1541 ATCTATTTCTCAAGCTTTCT 71 10494 10513 2999 567340 1523 1542 AATCTATTTCTCAAGCTTTC 74 10495 10514 3000 567341 1524 1543 AAATCTATTTCTCAAGCTTT 63 10496 10515 3001 567342 1525 1544 AAAATCTATTTCTCAAGCTT 54 10497 10516 3002 567343 1532 1551 GATAAAAAAAATCTATTTCT 30 10504 10523 3003 567344 1548 1567 TAGACAGTGACTTTAAGATA 37 10520 10539 3004 567345 1549 1568 ATAGACAGTGACTTTAAGAT 29 10521 10540 3005 567346 1550 1569 AATAGACAGTGACTTTAAGA 48 10522 10541 3006 567347 1551 1570 AAATAGACAGTGACTTTAAG 26 10523 10542 3007 567348 1552 1571 TAAATAGACAGTGACTTTAA 26 10524 10543 3008 567349 1553 1572 TTAAATAGACAGTGACTTTA 50 10525 10544 3009 567350 1554 1573 CTTAAATAGACAGTGACTTT 63 10526 10545 3010 567351 1555 1574 TCTTAAATAGACAGTGACTT 57 10527 10546 3011 567352 1556 1575 ATCTTAAATAGACAGTGACT 69 10528 10547 3012 567353 1557 1576 AATCTTAAATAGACAGTGAC 40 10529 10548 3013 567354 1558 1577 TAATCTTAAATAGACAGTGA 30 10530 10549 3014 567355 1559 1578 TTAATCTTAAATAGACAGTG 25 10531 10550 3015 567356 1560 1579 TTTAATCTTAAATAGACAGT 0 10532 10551 3016 567357 1561 1580 GTTTAATCTTAAATAGACAG 34 10533 10552 3017 567358 1562 1581 TGTTTAATCTTAAATAGACA 5 10534 10553 3018 567359 1563 1582 ATGTTTAATCTTAAATAGAC 0 10535 10554 3019 567360 1567 1586 TTGTATGTTTAATCTTAAAT 0 10539 10558 3020 567361 1568 1587 ATTGTATGTTTAATCTTAAA 8 10540 10559 3021 567362 1569 1588 GATTGTATGTTTAATCTTAA 20 10541 10560 3022 567363 1570 1589 TGATTGTATGTTTAATCTTA 29 10542 10561 3023 567364 1574 1593 TATGTGATTGTATGTTTAAT 7 10546 10565 3024 567365 1576 1595 GTTATGTGATTGTATGTTTA 43 10548 10567 3025 567366 1580 1599 TAAGGTTATGTGATTGTATG 28 10552 10571 3026 567367 1581 1600 TTAAGGTTATGTGATTGTAT 31 10553 10572 3027 567368 1585 1604 TTCTTTAAGGTTATGTGATT 12 10557 10576 3028

Example 117: Dose-Dependent Antisense Inhibition of Human ANGPTL3 in Hep3B Cells by MOE Gapmers

5-10-5 MOE gapmers from the studies described above exhibiting significant in vitro inhibition of ANGPTL3 mRNA were selected and tested at various doses in Hep3B cells. Cells were plated at a density of 20,000 cells per well and transfected using electroporation with 0.75 μM, 1.50 μM, 3.00 μM, 6.00 μM and 12.00 μM concentrations of antisense oligonucleotide, as specified in the Table below. After a treatment period of approximately 16 hours, RNA was isolated from the cells and ANGPTL3 mRNA levels were measured by quantitative real-time PCR. Human primer probe set RTS3492_MGB was used to measure mRNA levels. ANGPTL3 mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN. Results are presented as percent inhibition of ANGPTL3, relative to untreated control cells.

The half maximal inhibitory concentration (IC₅₀) of each oligonucleotide is also presented. ANGPTL3 mRNA levels were significantly reduced in a dose-dependent manner in antisense oligonucleotide treated cells.

TABLE 139 0.75 3.00 12.00 IC₅₀ SEQ ID ISIS No μM 1.50 μM μM 6.00 μM μM (μM) NO 233717 23 45 13 33 40 >12 14 544120 45 65 76 88 91 0.7 15 544145 38 42 61 82 84 1.6 16 544156 31 42 63 78 84 1.8 17 544162 35 43 71 76 82 1.6 18 544166 30 47 60 76 84 1.8 19 544199 54 61 73 83 84 0.5 20 544355 45 46 69 77 83 1.2 21 544368 12 37 63 74 81 2.6 22 544373 1 27 40 29 28 >12 23 544376 26 53 61 63 59 2.4 24 544380 16 33 41 64 39 8.4 25 544383 14 33 46 61 63 4.4 26 544410 10 41 48 62 69 3.6 27

Example 118: Dose-Dependent Antisense Inhibition of Human ANGPTL3 in Hep3B Cells by MOE Gapmers

5-10-5 MOE gapmers from the studies described above exhibiting significant in vitro inhibition of ANGPTL3 mRNA were selected and tested at various doses in Hep3B cells. Cells were plated at a density of 20,000 cells per well and transfected using electroporation with 0.813 μM, 1.625 μM, 3.25 μM, 6.50 μM and 13.00 μM concentrations of antisense oligonucleotide, as specified in the Table below. After a treatment period of approximately 16 hours, RNA was isolated from the cells and ANGPTL3 mRNA levels were measured by quantitative real-time PCR. Human primer probe set RTS3492_MGB was used to measure mRNA levels. ANGPTL3 mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of ANGPTL3, relative to untreated control cells.

The half maximal inhibitory concentration (IC₅₀) of each oligonucleotide is also presented. ANGPTL3 mRNA levels were significantly reduced in a dose-dependent manner in antisense oligonucleotide treated cells.

TABLE 140 SEQ 0.813 1.625 IC₅₀ ID ISIS No μM μM 3.25 μM 6.50 μM 13.00 μM (μM) NO 337487 17 37 58 72 92 2.7 28 337492 0 0 0 5 58 >13 29 544120 23 40 65 81 91 2.2 15 560236 39 22 46 9 60 >13 30 560265 38 48 58 64 73 2.0 31 560268 37 57 60 71 83 1.5 32 560285 5 29 48 68 78 3.8 33 560306 45 64 67 81 86 0.9 34 560400 48 63 75 87 88 0.7 35 560401 49 75 79 89 88 0.5 36 560402 42 67 70 85 90 0.9 37 560469 43 55 70 74 83 1.2 38 560470 31 54 64 73 81 1.8 39 560471 26 43 59 62 77 2.7 40 560474 42 50 60 54 72 1.8 41

TABLE 141 SEQ 0.813 1.625 IC₅₀ ID ISIS No μM μM 3.25 μM 6.50 μM 13.00 μM (μM) NO 337487 20 35 51 78 89 1.8 28 544120 31 46 62 84 90 0.5 15 544145 4 36 60 58 89 3.8 16 544156 22 35 46 66 73 1.8 17 544162 2 21 54 69 87 >13 18 544166 15 0 25 59 89 >13 19 544199 61 37 57 53 81 0.9 20 544355 0 47 50 73 84 >13 21 544376 4 14 38 66 88 0.9 24 560566 53 68 70 76 85 >13 42 560567 55 70 75 78 89 2.7 43 560574 49 63 68 74 84 2.0 44 560596 28 40 41 52 75 1.5 45 560607 35 53 65 70 85 3.8 46 560608 40 50 62 68 83 0.9 47 560723 36 51 59 65 75 2.2 48 560735 36 44 59 72 85 >13 49 560736 26 34 50 64 80 0.7 50 560744 28 49 59 75 83 0.9 51 560778 24 46 60 67 85 1.8 52 560789 14 23 36 49 71 2.7 53 560811 32 50 65 73 87 1.2 54 560856 0 20 17 32 69 3.8 55 560925 2 16 38 52 82 2.7 56 560936 0 0 24 41 65 0.5 57 560938 0 26 30 43 50 0.9 58 560942 0 0 12 36 74 1.8 59 560956 0 16 16 68 81 0.5 60

TABLE 142 IC₅₀ SEQ ID ISIS No 0.813 μM 1.625 μM 3.25 μM 6.50 μM 13.00 μM (μM) NO 337487 20 35 51 78 89 2.7 28 544120 31 46 62 84 90 1.9 15 560566 53 68 70 76 85 0.5 42 560567 55 70 75 78 89 0.4 43 560574 49 63 68 74 84 0.7 44 560596 28 40 41 52 75 3.9 45 560607 35 53 65 70 85 1.6 46 560608 40 50 62 68 83 1.6 47 560723 36 51 59 65 75 1.9 48 560735 36 44 59 72 85 2.0 49 560736 26 34 50 64 80 3.2 50 560744 28 49 59 75 83 2.1 51 560778 24 46 60 67 85 2.4 52 560789 14 23 36 49 71 5.7 53 560811 32 50 65 73 87 1.8 54

TABLE 143 IC₅₀ SEQ ID ISIS No 0.813 μM 1.625 μM 3.25 μM 6.50 μM 13.00 μM (μM) NO 337487 10 21 49 73 90 3.4 28 544120 19 38 62 77 88 2.5 15 560768 1 14 14 28 51 >13 61 560777 13 35 37 56 80 4.2 62 560791 13 28 28 24 11 >13 63 560794 8 31 42 57 76 4.4 64 560799 0 14 21 43 72 7.2 65 560803 26 44 52 55 69 3.4 66 560815 16 26 26 52 60 7.6 67 560817 0 0 11 18 37 >13 68 560847 37 52 56 68 87 1.8 69 560879 15 18 38 53 72 5.4 70 560880 0 8 21 38 71 8.0 71 560891 7 25 32 35 62 8.9 72 560895 11 10 0 5 48 >13 73

TABLE 144 IC₅₀ SEQ ID ISIS No 0.813 μM 1.625 μM 3.25 μM 6.50 μM 13.00 μM (μM) NO 337487 20 14 38 65 88 3.9 28 544120 22 34 51 71 86 2.9 15 544145 21 39 62 63 90 2.6 16 544156 31 41 55 72 78 2.4 17 544162 0 37 59 75 87 2.7 18 544166 8 43 45 55 75 4.0 19 544199 53 46 64 62 81 1.1 20 544355 0 0 52 72 84 2.9 21 544376 2 22 39 51 76 5.2 24 560856 10 29 36 41 69 6.4 55 560925 0 35 46 59 81 3.5 56 560936 18 9 35 55 69 5.9 57 560938 14 34 42 49 58 6.5 58 560942 8 13 27 47 77 6.1 59 560956 16 31 0 69 81 3.9 60

TABLE 145 IC₅₀ SEQ ID ISIS No 0.813 μM 1.625 μM 3.25 μM 6.50 μM 13.00 μM (μM) NO 233717 11 0 33 58 75 5.0 14 337484 39 54 55 66 79 1.7 74 337487 35 42 67 82 92 1.8 28 544120 53 47 78 84 92 <0.8 15 563523 12 44 59 63 79 3.0 75 563547 33 51 55 43 58 4.6 76 563580 61 73 71 82 91 <0.8 77 563637 36 55 69 77 88 1.4 78 563639 56 71 79 88 93 <0.8 79 563641 30 42 56 77 84 2.2 80 563669 28 61 66 79 85 1.6 81 563681 35 67 74 75 70 0.9 82 563682 41 45 68 76 85 1.5 83 567068 32 37 50 66 81 2.8 84 567069 23 28 48 56 62 5.0 85

TABLE 146 IC₅₀ SEQ ID ISIS No 0.813 μM 1.625 μM 3.25 μM 6.50 μM 13.00 μM (μM) NO 233717 9 0 25 62 74 5.5 14 337487 22 40 71 84 92 2.1 28 337516 36 54 78 81 92 1.3 86 544120 25 50 72 86 92 1.8 15 567078 54 64 70 78 78 <0.8 87 567115 55 65 72 80 81 <0.8 88 567134 33 58 53 57 69 2.2 89 567233 54 74 83 87 91 <0.8 90 567291 54 67 71 80 89 <0.8 91 567310 36 61 73 80 89 1.2 92 567320 63 77 88 88 92 <0.8 93 567321 55 75 89 89 93 <0.8 94 567330 31 68 76 85 93 1.2 95 567334 36 54 76 82 87 1.3 96 567335 31 49 72 80 92 1.7 97

TABLE 147 IC₅₀ SEQ ID ISIS No 0.813 μM 1.625 μM 3.25 μM 6.50 μM 13.00 μM (μM) NO 233717 0 0 23 66 64 6.6 14 337487 13 44 60 74 85 2.6 28 544120 24 47 53 78 83 2.3 15 568034 35 54 51 59 46 4.2 98 568047 36 55 70 69 72 1.4 99 568048 41 64 63 66 66 0.9 100 568049 50 70 70 74 73 <0.8 101 568056 33 56 68 63 64 1.7 102 568144 27 57 63 63 76 2.0 103 568146 50 61 61 63 77 <0.8 104 568151 23 46 59 68 66 2.8 105 568206 24 40 56 61 75 3.0 106 568293 0 39 46 59 78 4.1 107 568300 22 36 61 68 73 3.0 108 568330 16 48 54 73 82 2.7 109

Example 119: Antisense Inhibition of Human ANGPTL3 in Hep3B Cells by Deoxy, MOE and (S)-cEt Gapmers

Additional antisense oligonucleotides were designed targeting an ANGPTL3 nucleic acid and were tested for their effects on ANGPTL3 mRNA in vitro. Cultured Hep3B cells at a density of 20,000 cells per well were transfected using electroporation with 4,500 nM of antisense oligonucleotide. After a treatment period of approximately 24 hours, RNA was isolated from the cells and ANGPTL3 mRNA levels were measured by quantitative real-time PCR. Human primer probe set RTS3492_MGB was used to measure mRNA levels. ANGPTL3 mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of ANGPTL3, relative to untreated control cells.

The newly designed chimeric antisense oligonucleotides in the Tables below were designed as deoxy, MOE, and (S)-cEt oligonucleotides. The deoxy, MOE and (S)-cEt oligonucleotides are 16 nucleosides in length wherein the nucleoside have either a MOE sugar modification, a (S)-cEt sugar modification, or a deoxy sugar residue. The sugar modifications of each antisense oligonucleotide is described as ‘eek-d10-kke’, where ‘k’ indicates a (S)-cEt sugar modification; ‘d’ indicates deoxyribose; the number indicates the number of deoxyribose sugars residues; and ‘e’ indicates a MOE sugar modification. The internucleoside linkages throughout each oligonucleotide are phosphorothioate (P═S) linkages. All cytosine residues throughout each oligonucleotide are 5-methylcytosines. “Start site” indicates the 5′-most nucleoside to which the oligonucleotide is targeted in the human gene sequence. “Stop site” indicates the 3′-most nucleoside to which the oligonucleotide is targeted human gene sequence. Each oligonucleotide listed in the Tables below is targeted to either the human ANGPTL3 mRNA, designated herein as SEQ ID NO: 1 (GENBANK Accession No. NM_014495.2) or the human ANGPTL3 genomic sequence, designated herein as SEQ ID NO: 2 (GENBANK Accession No. NT_032977.9 truncated from nucleotides 33032001 to 33046000). ‘n/a’ indicates that the antisense oligonucleotide does not target that particular gene sequence with 100% complementarity.

TABLE 148 Inhibition of ANGPTL3 mRNA by deoxy, MOE and cEt oligonucleotides targeting SEQ ID NO: 1 and 2 SEQ SEQ SEQ SEQ ID ID ID ID NO: 1 NO: NO: 2 NO: 2 SEQ ISIS Start 1 Stop % Start Stop ID NO Site Site Sequence inhibition Site Site NO 561681 N/A N/A TCTGGAAGCAGACCTA 37 3096 3111 3029 561682 N/A N/A CTTCTGGAAGCAGACC 27 3098 3113 3030 561683 N/A N/A AAATAAGGTATAGTGA 2 11084 11099 3031 561684 N/A N/A TAGTATTAAGTGTTAA 14 11133 11148 3032 561685 N/A N/A TCATAGTATTAAGTGT 0 11136 11151 3033 561686 N/A N/A AGATTCCTTTACAATT 21 11160 11175 3034 561687 N/A N/A ACAAGATTCCTTTACA 21 11163 11178 3035 561688 N/A N/A CTGACAAGATTCCTTT 70 11166 11181 3036 561689 N/A N/A AATCTGACAAGATTCC 83 11169 11184 180 561690 N/A N/A TGTAATCTGACAAGAT 46 11172 11187 3037 561691 N/A N/A TACTGTAATCTGACAA 47 11175 11190 3038 561692 N/A N/A TCTTACTGTAATCTGA 50 11178 11193 3039 561693 N/A N/A CATTCTTACTGTAATC 40 11181 11196 3040 561694 N/A N/A GTTCATTCTTACTGTA 71 11184 11199 3041 561695 N/A N/A ATATGTTCATTCTTAC 2 11188 11203 3042 561696 N/A N/A GCCACAAATATGTTCA 80 11195 11210 3043 561697 N/A N/A GATGCCACAAATATGT 70 11198 11213 3044 561698 N/A N/A CTCGATGCCACAAATA 80 11201 11216 181 561699 N/A N/A TAACTCGATGCCACAA 86 11204 11219 182 561700 N/A N/A CTTTAACTCGATGCCA 77 11207 11222 3045 561701 N/A N/A AAACTTTAACTCGATG 39 11210 11225 3046 561702 N/A N/A TATAAACTTTAACTCG 13 11213 11228 3047 561703 N/A N/A CACAGCATATTTAGGG 71 11233 11248 3048 561704 N/A N/A TAGAATCACAGCATAT 68 11239 11254 3049 561705 N/A N/A TATTAGAATCACAGCA 73 11242 11257 3050 561706 N/A N/A AATGTATTAGAATCAC 40 11246 11261 3051 561707 N/A N/A ACGAATGTATTAGAAT 22 11249 11264 3052 561708 N/A N/A TACACGAATGTATTAG 33 11252 11267 3053 561709 N/A N/A ACCTACACGAATGTAT 42 11255 11270 3054 561710 N/A N/A AAAACCTACACGAATG 24 11258 11273 3055 561711 N/A N/A TTGAAAACCTACACGA 34 11261 11276 3056 561712 N/A N/A TACTTGAAAACCTACA 33 11264 11279 3057 561713 N/A N/A GTTTATTTCTACTTGA 53 11273 11288 3058 561714 N/A N/A GAGGTTTATTTCTACT 69 11276 11291 3059 561715 N/A N/A TACGAGGTTTATTTCT 21 11279 11294 3060 561716 N/A N/A TGTTACGAGGTTTATT 47 11282 11297 3061 561717 N/A N/A ACTTGTTACGAGGTTT 70 11285 11300 3062 561718 N/A N/A CAGTAACTTGTTACGA 60 11290 11305 3063 561719 N/A N/A GTTCAGTAACTTGTTA 40 11293 11308 3064 561720 N/A N/A TCAGGCTGTTTAAACG 59 11308 11323 3065 561721 N/A N/A TTGTCAGGCTGTTTAA 74 11311 11326 3066 561722 N/A N/A TGCTTGTCAGGCTGTT 82 11314 11329 183 561723 N/A N/A ACATGCTTGTCAGGCT 84 11317 11332 184 561724 N/A N/A TATACATGCTTGTCAG 75 11320 11335 3067 561725 N/A N/A GTCTTTGTTTATTGAA 49 11347 11362 3068 561726 N/A N/A TGGGTCTTTGTTTATT 27 11350 11365 3069 561727 N/A N/A GACTGGGTCTTTGTTT 20 11353 11368 3070 561728 N/A N/A ATAATTTAGGGACTGG 20 11363 11378 3071 561729 N/A N/A TCTATAATTTAGGGAC 39 11366 11381 3072 561730 N/A N/A CGATAAACATGCAAGA 68 11394 11409 3073 561731 N/A N/A TGTCGATAAACATGCA 80 11397 11412 3074 561732 N/A N/A TGATGTCGATAAACAT 68 11400 11415 3075 561733 N/A N/A TTGTGATGTCGATAAA 28 11403 11418 3076 561734 N/A N/A CTGTTGTGATGTCGAT 74 11406 11421 3077 561735 N/A N/A GATCTGTTGTGATGTC 59 11409 11424 3078 561736 N/A N/A AGGGATCTGTTGTGAT 24 11412 11427 3079 561737 N/A N/A TTTAGGGATCTGTTGT 19 11415 11430 3080 561738 N/A N/A GGATTTAGGGATCTGT 27 11418 11433 3081 561739 N/A N/A GATTTAGGGATTTAGG 44 11425 11440 3082 561740 N/A N/A TCTTTAGGGATTTAGG 38 11433 11448 3083 561741 N/A N/A TAATCTTTAGGGATTT 0 11436 11451 3084 561742 N/A N/A ATCTAATCTTTAGGGA 0 11439 11454 3085 561743 N/A N/A TGTATCTAATCTTTAG 15 11442 11457 3086 561744 N/A N/A AAATTTGTATCTAATC 21 11447 11462 3087 561745 N/A N/A GTAAAAAATTTGTATC 23 11452 11467 3088 561746 N/A N/A GTGGTAAAAAATTTGT 32 11455 11470 3089 561747 N/A N/A GATACTGTGGTAAAAA 45 11461 11476 3090 561748 N/A N/A AGTGATACTGTGGTAA 60 11464 11479 3091 561749 N/A N/A ACAAGTGATACTGTGG 75 11467 11482 3092 561750 N/A N/A CTGACAAGTGATACTG 59 11470 11485 3093 561751 N/A N/A ATTCTGACAAGTGATA 48 11473 11488 3094 561752 N/A N/A TAAATTCTGACAAGTG 59 11476 11491 3095 561753 N/A N/A TACTGGCAGTTTTAAA 42 11508 11523 3096 561754 N/A N/A TCTTACTGGCAGTTTT 51 11511 11526 3097 561755 N/A N/A ATTTCTTACTGGCAGT 69 11514 11529 3098 561756 N/A N/A AAAATTTCTTACTGGC 57 11517 11532 3099 561757 N/A N/A AACAAATGGGTTTAAT 0 11535 11550 3100 562374 N/A N/A GAATATTTGCAAGTCT 68 9230 9245 3101 562375 N/A N/A GTAGAGGAATATTTGC 83 9236 9251 151 562376 N/A N/A TCATTGGTAGAGGAAT 23 9242 9257 3102 562377 N/A N/A ATATTTTAAAGTCTCG 17 9258 9273 3103 562378 N/A N/A GTTACATTATTATAGA 29 9273 9288 3104 562379 N/A N/A GTGAAATGTGTTACAT 54 9282 9297 3105 562380 N/A N/A TCACCAGTGAAATGTG 64 9288 9303 3106 562381 N/A N/A CATGTTTCACCAGTGA 78 9294 9309 3107 562382 N/A N/A ACAAGACATGTTTCAC 36 9300 9315 3108 562383 N/A N/A CATATGACAAGACATG 42 9306 9321 3109 562384 N/A N/A CTATAATGCATATGAC 5 9314 9329 3110 562385 N/A N/A TCCTTTCTATAATGCA 65 9320 9335 3111 562386 N/A N/A TGATTATCCTTTCTAT 27 9326 9341 3112 562387 N/A N/A AAAGTCTGATTATCCT 90 9332 9347 152 562388 N/A N/A TAACTGAAAGTCTGAT 59 9338 9353 3113 562389 N/A N/A GTGCACAAAAATGTTA 42 9366 9381 3114 562390 N/A N/A AGCTATGTGCACAAAA 77 9372 9387 3115 562391 N/A N/A GAAGATAGCTATGTGC 64 9378 9393 3116 562392 N/A N/A TTTATTGAAGATAGCT 33 9384 9399 3117 562393 N/A N/A TCATTTTAGTGTATCT 40 9424 9439 3118 562394 N/A N/A CCTTGATCATTTTAGT 15 9430 9445 3119 562395 N/A N/A TGAATCCCTTGATCAT 59 9436 9451 3120 562396 N/A N/A TAGTCTTGAATCCCTT 83 9442 9457 153 562397 N/A N/A GTTGTTTAGTCTTGAA 65 9448 9463 3121 562398 N/A N/A AATTGAGTTGTTTAGT 21 9454 9469 3122 562399 N/A N/A GCAACTAATTGAGTTG 15 9460 9475 3123 562400 N/A N/A ATTGGTGCAACTAATT 25 9466 9481 3124 562401 N/A N/A GTTTTTTATTGGTGCA 53 9473 9488 3125 562402 N/A N/A GGACACTGACAGTTTT 43 9496 9511 3126 562403 N/A N/A CAGGTTGGACACTGAC 23 9502 9517 3127 562404 N/A N/A TAAGTACAGGTTGGAC 33 9508 9523 3128 562405 N/A N/A AGTTATTAAGTACAGG 34 9514 9529 3129 562406 N/A N/A TCTGTGAGTTATTAAG 10 9520 9535 3130 562407 N/A N/A ACCAAAATTCTCCTGA 1 9554 9569 3131 562408 N/A N/A ACCTGAATAACCCTCT 73 9811 9826 3132 562409 N/A N/A GGTATCAGAAAAAGAT 14 9827 9842 3133 562410 N/A N/A AGTATTGGTATCAGAA 13 9833 9848 3134 562411 N/A N/A GGAAGATACTTTGAAG 25 9861 9876 3135 562412 N/A N/A AATGTGGGAAGATACT 23 9867 9882 3136 562413 N/A N/A CAGATAATAGCTAATA 29 9882 9897 3137 562414 N/A N/A TCATTGCAGATAATAG 45 9888 9903 3138 562415 N/A N/A AAGTTGTCATTGCAGA 86 9894 9909 154 562416 N/A N/A GATTCGGATTTTTAAA 19 9909 9924 3139 562417 N/A N/A ATTTGGGATTCGGATT 34 9915 9930 3140 562418 N/A N/A ACGCTTATTTGGGATT 64 9921 9936 3141 562419 N/A N/A TCTAGAGAGAAAACGC 64 9933 9948 3142 562420 N/A N/A AGTTAAGAGGTTTTCG 34 9949 9964 3143 562421 N/A N/A CATTATAGTTAAGAGG 24 9955 9970 3144 562422 N/A N/A CACTTTCATTATAGTT 13 9961 9976 3145 562423 N/A N/A TAGAATGAACACTTTC 63 9970 9985 3146 562424 N/A N/A TTGAACTAGAATGAAC 16 9976 9991 3147 562425 N/A N/A ACCTGATTGAACTAGA 51 9982 9997 3148 562426 N/A N/A TAAAATACCTGATTGA 19 9988 10003 3149 562427 N/A N/A TAGAGGTAAAATACCT 12 9994 10009 3150 562428 N/A N/A GAAGATTAGAGGTAAA 1 10000 10015 3151 562429 N/A N/A TCTGAGGAAGATTAGA 31 10006 10021 3152 562430 N/A N/A TATACACTACCAAAAA 0 10030 10045 3153 562431 N/A N/A ATAATCTATACACTAC 0 10036 10051 3154 562432 N/A N/A TAAGTCCCAATTTTAA 33 10065 10080 3155 562433 N/A N/A TCTGTATAAGTCCCAA 89 10071 10086 155 562434 N/A N/A CCAGTTTTAAATAATC 20 10085 10100 3156 562435 N/A N/A TGTATCCCAGTTTTAA 44 10091 10106 3157 562436 N/A N/A GATGCATGTATCCCAG 91 10097 10112 156 562437 N/A N/A GTTTTAGATGCATGTA 69 10103 10118 3158 562438 N/A N/A TACAGTGTTTTAGATG 28 10109 10124 3159 562439 N/A N/A GTAAGTTTATCTTCCT 78 10138 10153 157 562440 N/A N/A TTCCCCGTAAGTTTAT 33 10144 10159 3160 562441 N/A N/A CTGTATTTCCCCGTAA 55 10150 10165 3161 562442 N/A N/A CTGTTACTGTATTTCC 79 10156 10171 158 562443 N/A N/A TAGTTACTGTTACTGT 70 10162 10177 3162 562444 N/A N/A CGTATGTAGTTACTGT 66 10168 10183 3163 562445 N/A N/A AATGGGTACAGACTCG 72 10182 10197 3164 562446 N/A N/A GCAATTTAATGGGTAC 59 10189 10204 3165 562447 N/A N/A GATAGATATGCAATTT 20 10198 10213 3166 562448 N/A N/A AAAGGAGATAGATATG 22 10204 10219 3167 562449 N/A N/A CCTCCTAAAGGAGATA 42 10210 10225 3168 562450 N/A N/A CACCAGCCTCCTAAAG 37 10216 10231 3169 560990 709 724 TTCTTGGTGCTCTTGG 89 6722 6737 111 561373 1197 1212 TTTGTGATCCCAAGTA 40 9772 9787 3170 561374 1199 1214 GCTTTGTGATCCCAAG 76 9774 9789 3171 561375 1201 1216 TTGCTTTGTGATCCCA 82 9776 9791 3172 561376 1203 1218 TTTTGCTTTGTGATCC 40 9778 9793 3173 561377 1205 1220 CCTTTTGCTTTGTGAT 38 9780 9795 3174 561378 1207 1222 GTCCTTTTGCTTTGTG 75 9782 9797 3175 561379 1209 1224 GTGTCCTTTTGCTTTG 40 9784 9799 3176 561527 1604 1619 GAAATGTAAACGGTAT 47 10576 10591 3177 561528 1606 1621 GAGAAATGTAAACGGT 89 10578 10593 174 561529 1608 1623 TTGAGAAATGTAAACG 55 10580 10595 3178 561530 1611 1626 TGATTGAGAAATGTAA 18 10583 10598 3179 561531 1613 1628 TTTGATTGAGAAATGT 30 10585 10600 3180 561532 1619 1634 AAGAATTTTGATTGAG 53 10591 10606 3181 561533 1621 1636 ATAAGAATTTTGATTG 29 10593 10608 3182 561534 1632 1647 CAAATAGTATTATAAG 6 10604 10619 3183 561535 1653 1668 CCCACATCACAAAATT 70 10625 10640 3184 561536 1657 1672 GATTCCCACATCACAA 77 10629 10644 3185 561537 1659 1674 TTGATTCCCACATCAC 78 10631 10646 3186 561538 1661 1676 AATTGATTCCCACATC 68 10633 10648 3187 561539 1663 1678 AAAATTGATTCCCACA 72 10635 10650 3188 561540 1665 1680 CTAAAATTGATTCCCA 54 10637 10652 3189 561541 1668 1683 CATCTAAAATTGATTC 0 10640 10655 3190 561542 1670 1685 ACCATCTAAAATTGAT 35 10642 10657 3191 561543 1672 1687 TGACCATCTAAAATTG 55 10644 10659 3192 561544 1674 1689 TGTGACCATCTAAAAT 56 10646 10661 3193 561545 1676 1691 ATTGTGACCATCTAAA 73 10648 10663 3194 561546 1678 1693 AGATTGTGACCATCTA 67 10650 10665 3195 561547 1680 1695 CTAGATTGTGACCATC 50 10652 10667 3196 561548 1682 1697 ATCTAGATTGTGACCA 77 10654 10669 3197 561549 1684 1699 TAATCTAGATTGTGAC 55 10656 10671 3198 561550 1686 1701 TATAATCTAGATTGTG 28 10658 10673 3199 561551 1688 1703 ATTATAATCTAGATTG 52 10660 10675 3200 561552 1690 1705 TGATTATAATCTAGAT 43 10662 10677 3201 561553 1692 1707 ATTGATTATAATCTAG 53 10664 10679 3202 561554 1694 1709 CTATTGATTATAATCT 54 10666 10681 3203 561555 1696 1711 ACCTATTGATTATAAT 44 10668 10683 3204 561556 1698 1713 TCACCTATTGATTATA 52 10670 10685 3205 561557 1700 1715 GTTCACCTATTGATTA 50 10672 10687 3206 561558 1702 1717 AAGTTCACCTATTGAT 58 10674 10689 3207 561559 1704 1719 ATAAGTTCACCTATTG 66 10676 10691 3208 561560 1706 1721 TAATAAGTTCACCTAT 38 10678 10693 3209 561561 1708 1723 TTTAATAAGTTCACCT 50 10680 10695 3210 561562 1710 1725 TATTTAATAAGTTCAC 32 10682 10697 3211 561563 1712 1727 GTTATTTAATAAGTTC 47 10684 10699 3212 561564 1761 1776 CATATGATGCCTTTTA 63 10733 10748 3213 561565 1763 1778 CTCATATGATGCCTTT 81 10735 10750 175 561566 1765 1780 AGCTCATATGATGCCT 81 10737 10752 176 561567 1767 1782 TTAGCTCATATGATGC 84 10739 10754 177 561568 1769 1784 TATTAGCTCATATGAT 46 10741 10756 3214 561569 1771 1786 GATATTAGCTCATATG 49 10743 10758 3215 561570 1773 1788 GTGATATTAGCTCATA 81 10745 10760 3216 561571 1775 1790 TTGTGATATTAGCTCA 85 10747 10762 178 561572 1777 1792 AGTTGTGATATTAGCT 68 10749 10764 3217 561573 1779 1794 AAAGTTGTGATATTAG 45 10751 10766 3218 561574 1781 1796 GGAAAGTTGTGATATT 27 10753 10768 3219 561575 1783 1798 TGGGAAAGTTGTGATA 36 10755 10770 3220 561576 1785 1800 ACTGGGAAAGTTGTGA 83 10757 10772 179 561577 1787 1802 AAACTGGGAAAGTTGT 56 10759 10774 3221 561578 1789 1804 TTAAACTGGGAAAGTT 44 10761 10776 3222 561579 1794 1809 GTTTTTTAAACTGGGA 58 10766 10781 3223 561580 1796 1811 TAGTTTTTTAAACTGG 0 10768 10783 3224 561581 1802 1817 GAGTACTAGTTTTTTA 18 10774 10789 3225 561582 1804 1819 AAGAGTACTAGTTTTT 55 10776 10791 3226 561583 1806 1821 ACAAGAGTACTAGTTT 51 10778 10793 3227 561584 1808 1823 TAACAAGAGTACTAGT 53 10780 10795 3228 561585 1810 1825 TTTAACAAGAGTACTA 48 10782 10797 3229 561586 1812 1827 GTTTTAACAAGAGTAC 49 10784 10799 3230 561587 1814 1829 GAGTTTTAACAAGAGT 54 10786 10801 3231 561588 1816 1831 TAGAGTTTTAACAAGA 9 10788 10803 3232 561589 1819 1834 GTTTAGAGTTTTAACA 24 10791 10806 3233 561590 1822 1837 CAAGTTTAGAGTTTTA 30 10794 10809 3234 561591 1824 1839 GTCAAGTTTAGAGTTT 60 10796 10811 3235 561592 1826 1841 TAGTCAAGTTTAGAGT 56 10798 10813 3236 561593 1828 1843 TTTAGTCAAGTTTAGA 41 10800 10815 3237 561594 1830 1845 TATTTAGTCAAGTTTA 14 10802 10817 3238 561595 1832 1847 TGTATTTAGTCAAGTT 39 10804 10819 3239 561596 1834 1849 TCTGTATTTAGTCAAG 51 10806 10821 3240 561597 1836 1851 CCTCTGTATTTAGTCA 72 10808 10823 3241 561598 1838 1853 GTCCTCTGTATTTAGT 55 10810 10825 3242 561599 1840 1855 CAGTCCTCTGTATTTA 63 10812 10827 3243 561600 1842 1857 ACCAGTCCTCTGTATT 66 10814 10829 3244 561601 1844 1859 TTACCAGTCCTCTGTA 57 10816 10831 3245 561602 1846 1861 AATTACCAGTCCTCTG 43 10818 10833 3246 561603 1848 1863 ACAATTACCAGTCCTC 67 10820 10835 3247

TABLE 149 Inhibition of ANGPTL3 mRNA by deoxy, MOE and (S)-cEt gapmers targeting SEQ ID NO: 1 and 2 SEQ SEQ SEQ ID SEQ ID ID NO: NO: ID NO: NO: 2 SEQ 1 Start 1 Stop % 2 Start Stop ID ISIS NO Site Site Sequence inhibition Site Site NO 561770 N/A N/A ACAAAGGTAGGTCACC 77 11576 11591 143 586719 N/A N/A TCTGACAAGATTCCTT 76 11167 11182 3248 586720 N/A N/A ATCTGACAAGATTCCT 79 11168 11183 3249 586721 N/A N/A TAATCTGACAAGATTC 50 11170 11185 3250 586722 N/A N/A GTAATCTGACAAGATT 41 11171 11186 3251 586723 N/A N/A CTTGTCAGGCTGTTTA 50 11312 11327 3252 586724 N/A N/A GCTTGTCAGGCTGTTT 81 11313 11328 3253 586725 N/A N/A ATGCTTGTCAGGCTGT 78 11315 11330 3254 586726 N/A N/A TACATGCTTGTCAGGC 78 11318 11333 3255 586727 N/A N/A ATACATGCTTGTCAGG 76 11319 11334 3256 586728 N/A N/A AAAGGTAGGTCACCAT 72 11574 11589 3257 586729 N/A N/A CAAAGGTAGGTCACCA 69 11575 11590 3258 586730 N/A N/A GACAAAGGTAGGTCAC 55 11577 11592 3259 586731 N/A N/A TGACAAAGGTAGGTCA 32 11578 11593 3260 586732 N/A N/A TCTGACATAGCTTTTT 63 5436 5451 3261 586733 N/A N/A ATTCTGACATAGCTTT 76 5438 5453 3262 586734 N/A N/A GATTCTGACATAGCTT 73 5439 5454 3263 586735 N/A N/A GGATTCTGACATAGCT 81 5440 5455 3264 586736 N/A N/A ATGGATTCTGACATAG 74 5442 5457 3265 586737 N/A N/A CATGGATTCTGACATA 72 5443 5458 3266 586738 N/A N/A ACATGGATTCTGACAT 59 5444 5459 3267 586739 N/A N/A TACATGGATTCTGACA 71 5445 5460 3268 586740 N/A N/A ATACATGGATTCTGAC 60 5446 5461 3269 586741 N/A N/A TTTAGCAGCACTACTA 65 5628 5643 3270 586742 N/A N/A TTTTAGCAGCACTACT 51 5629 5644 3271 586743 N/A N/A CTTTTAGCAGCACTAC 74 5630 5645 3272 586744 N/A N/A CCTTTTAGCAGCACTA 83 5631 5646 223 586745 N/A N/A ACCTTTTAGCAGCACT 84 5632 5647 224 586746 N/A N/A AAACCTTTTAGCAGCA 87 5634 5649 225 586747 N/A N/A AAAACCTTTTAGCAGC 80 5635 5650 3273 586748 N/A N/A GATAAAAAACCTTTTA 16 5640 5655 3274 586749 N/A N/A TGATAAAAAACCTTTT 25 5641 5656 3275 586750 N/A N/A AGATGTTGGCAGGTTG 72 6188 6203 3276 586751 N/A N/A TAGATGTTGGCAGGTT 76 6189 6204 3277 586752 N/A N/A GTAGATGTTGGCAGGT 73 6190 6205 3278 586753 N/A N/A TGTAGATGTTGGCAGG 65 6191 6206 3279 586754 N/A N/A CTGTAGATGTTGGCAG 61 6192 6207 3280 586755 N/A N/A ATCTGTAGATGTTGGC 84 6194 6209 226 586756 N/A N/A TATCTGTAGATGTTGG 71 6195 6210 3281 586757 N/A N/A ATATCTGTAGATGTTG 61 6196 6211 3282 586758 N/A N/A CATATCTGTAGATGTT 63 6197 6212 3283 586759 N/A N/A TTTGAACCAGGCTTTC 47 6243 6258 3284 586760 N/A N/A AATTTGAACCAGGCTT 78 6245 6260 3285 586761 N/A N/A TAATTTGAACCAGGCT 83 6246 6261 227 586762 N/A N/A CATAATTTGAACCAGG 81 6248 6263 3286 586763 N/A N/A ACATAATTTGAACCAG 36 6249 6264 3287 586764 N/A N/A TACATAATTTGAACCA 38 6250 6265 3288 586765 N/A N/A ATACATAATTTGAACC 15 6251 6266 3289 586766 N/A N/A ACATTGGTCGGAAAAC 43 6424 6439 3290 586767 N/A N/A GACATTGGTCGGAAAA 49 6425 6440 3291 586768 N/A N/A AGACATTGGTCGGAAA 59 6426 6441 3292 586769 N/A N/A CAGACATTGGTCGGAA 66 6427 6442 3293 586770 N/A N/A GCAGACATTGGTCGGA 80 6428 6443 3294 586771 N/A N/A AAGCAGACATTGGTCG 65 6430 6445 3295 586772 N/A N/A TGTACAGATTACCTGT 51 6506 6521 3296 586773 N/A N/A TTGTACAGATTACCTG 34 6507 6522 3297 586774 N/A N/A ATTGTACAGATTACCT 62 6508 6523 3298 586775 N/A N/A GATTGTACAGATTACC 59 6509 6524 3299 586776 N/A N/A AGATTGTACAGATTAC 46 6510 6525 3300 586777 N/A N/A TCAGATTGTACAGATT 63 6512 6527 3301 586778 N/A N/A TTCAGATTGTACAGAT 63 6513 6528 3302 586779 N/A N/A ATTCAGATTGTACAGA 71 6514 6529 3303 586780 N/A N/A TATTCAGATTGTACAG 55 6515 6530 3304 586781 N/A N/A TTATTCAGATTGTACA 52 6516 6531 3305 586782 N/A N/A TAGGTATGTCTTTTAT 52 6936 6951 3306 586783 N/A N/A TGTCTTAGGTATGTCT 76 6941 6956 3307 586784 N/A N/A ATTGTCTTAGGTATGT 73 6943 6958 3308 586785 N/A N/A GATTGTCTTAGGTATG 60 6944 6959 3309 586786 N/A N/A TTCTTAGATGGCGTGT 74 7207 7222 3310 586787 N/A N/A TTTTCTTAGATGGCGT 86 7209 7224 228 586788 N/A N/A ATTTTTCTTAGATGGC 75 7211 7226 3311 586789 N/A N/A CATTTTTCTTAGATGG 49 7212 7227 3312 586790 N/A N/A GCATTTTTCTTAGATG 47 7213 7228 3313 586791 N/A N/A ATAAGTCCCAATTTTA 27 10066 10081 3314 586792 N/A N/A TATAAGTCCCAATTTT 27 10067 10082 3315 586793 N/A N/A GTATAAGTCCCAATTT 28 10068 10083 3316 586794 N/A N/A TGTATAAGTCCCAATT 38 10069 10084 3317 586795 N/A N/A CTGTATAAGTCCCAAT 69 10070 10085 3318 586796 N/A N/A ATCTGTATAAGTCCCA 88 10072 10087 229 586797 N/A N/A AATCTGTATAAGTCCC 84 10073 10088 230 586798 N/A N/A TAATCTGTATAAGTCC 58 10074 10089 3319 586799 N/A N/A ATAATCTGTATAAGTC 21 10075 10090 3320 586800 N/A N/A AATAATCTGTATAAGT 12 10076 10091 3321 586801 N/A N/A TGCATGTATCCCAGTT 80 10095 10110 3322 586802 N/A N/A ATGCATGTATCCCAGT 83 10096 10111 231 586803 N/A N/A AGATGCATGTATCCCA 79 10098 10113 232 586804 N/A N/A TAGATGCATGTATCCC 87 10099 10114 3323 586805 N/A N/A TTAGATGCATGTATCC 78 10100 10115 3324 586806 N/A N/A TTTAGATGCATGTATC 50 10101 10116 3325 586653 7 22 GTGGAACTGTTTTCTT 63 3111 3126 3326 586656 9 24 ACGTGGAACTGTTTTC 72 3113 3128 3327 586658 99 114 TTGATCAATTCTGGAG 74 3203 3218 3328 586660 101 116 TCTTGATCAATTCTGG 71 3205 3220 3329 561011 102 117 GTCTTGATCAATTCTG 91 3206 3221 114 586661 103 118 TGTCTTGATCAATTCT 85 3207 3222 209 586663 134 149 GGCTCTGGAGATAGAG 63 3238 3253 3330 586665 136 151 TTGGCTCTGGAGATAG 63 3240 3255 3331 586668 140 155 GATTTTGGCTCTGGAG 64 3244 3259 3332 586669 142 157 TTGATTTTGGCTCTGG 89 3246 3261 210 561026 143 158 CTTGATTTTGGCTCTG 84 3247 3262 117 586670 144 159 TCTTGATTTTGGCTCT 71 3248 3263 3333 586671 146 161 AATCTTGATTTTGGCT 70 3250 3265 3334 586672 148 163 CAAATCTTGATTTTGG 81 3252 3267 3335 586673 298 313 GCAGCGATAGATCATA 76 3402 3417 3336 586674 300 315 TTGCAGCGATAGATCA 76 3404 3419 3337 586675 304 319 TGGTTTGCAGCGATAG 82 3408 3423 3338 586676 306 321 ACTGGTTTGCAGCGAT 89 3410 3425 211 586677 315 330 TTTGATTTCACTGGTT 62 3419 3434 3339 586678 317 332 TCTTTGATTTCACTGG 66 3421 3436 3340 586679 342 357 AGTTCTTCTCAGTTCC 77 3446 3461 3341 586680 476 491 TTAGTTAGTTGCTCTT 65 3580 3595 3342 586681 478 493 AGTTAGTTAGTTGCTC 69 3582 3597 3343 586682 703 718 GTGCTCTTGGCTTGGA 78 6716 6731 3344 586683 705 720 TGGTGCTCTTGGCTTG 77 6718 6733 3345 586684 802 817 TATGTTCACCTCTGTT 55 7387 7402 3346 586685 804 819 TGTATGTTCACCTCTG 79 7389 7404 3347 586686 1260 1275 ACACTCATCATGCCAC 72 10232 10247 3348 586687 1262 1277 CCACACTCATCATGCC 82 10234 10249 3349 586688 1308 1323 AGATTTTGCTCTTGGT 87 10280 10295 212 586689 1310 1325 TTAGATTTTGCTCTTG 78 10282 10297 3350 586690 1351 1366 CATTTTGAGACTTCCA 91 10323 10338 213 586691 1353 1368 TCCATTTTGAGACTTC 86 10325 10340 214 586692 1365 1380 AGAGTATAACCTTCCA 88 10337 10352 220 586693 1367 1382 ATAGAGTATAACCTTC 69 10339 10354 3351 586694 1402 1417 AATCTGTTGGATGGAT 59 10374 10389 3352 586695 1404 1419 TGAATCTGTTGGATGG 79 10376 10391 3353 586696 1420 1435 TTCATTCAAAGCTTTC 82 10392 10407 3354 586697 1422 1437 AGTTCATTCAAAGCTT 73 10394 10409 3355 561463 1423 1438 CAGTTCATTCAAAGCT 88 10395 10410 127 586698 1424 1439 TCAGTTCATTCAAAGC 69 10396 10411 3356 586699 1488 1503 GATTATTAGACCACAT 63 10460 10475 3357 586700 1490 1505 CAGATTATTAGACCAC 90 10462 10477 221 561487 1491 1506 CCAGATTATTAGACCA 95 10463 10478 131 586701 1492 1507 ACCAGATTATTAGACC 85 10464 10479 215 586702 1552 1567 TAGACAGTGACTTTAA 83 10524 10539 216 586703 1554 1569 AATAGACAGTGACTTT 70 10526 10541 3358 586704 1605 1620 AGAAATGTAAACGGTA 76 10577 10592 3359 586705 1607 1622 TGAGAAATGTAAACGG 83 10579 10594 217 586706 1762 1777 TCATATGATGCCTTTT 69 10734 10749 3360 586707 1764 1779 GCTCATATGATGCCTT 84 10736 10751 218 586708 1766 1781 TAGCTCATATGATGCC 83 10738 10753 222 561567 1767 1782 TTAGCTCATATGATGC 81 10739 10754 177 586709 1768 1783 ATTAGCTCATATGATG 40 10740 10755 3361 586710 1774 1789 TGTGATATTAGCTCAT 73 10746 10761 3362 586711 1776 1791 GTTGTGATATTAGCTC 80 10748 10763 3363 586712 1905 1920 TACTCTGTGCTGACGA 81 10877 10892 3364 586713 1907 1922 CATACTCTGTGCTGAC 81 10879 10894 3365 586714 2052 2067 GTTTAAAGACAGCGAA 72 11024 11039 3366 586715 2054 2069 TTGTTTAAAGACAGCG 81 11026 11041 3367 586716 2068 2083 GTAGTCATCTCCATTT 63 11040 11055 3368 586717 2070 2085 TAGTAGTCATCTCCAT 74 11042 11057 3369 561650 2071 2086 TTAGTAGTCATCTCCA 79 11043 11058 142 586718 2072 2087 CTTAGTAGTCATCTCC 84 11044 11059 219

Example 120: Antisense Inhibition of Human ANGPTL3 in Hep3B Cells by Deoxy, MOE and (S)-cEt Gapmers

Additional antisense oligonucleotides were designed targeting an ANGPTL3 nucleic acid and were tested for their effects on ANGPTL3 mRNA in vitro. ISIS 337487 and ISIS 233717, which are 5-10-5 MOE gapmers, were also included in the assay as benchmark oligonucleotides. Cultured Hep3B cells at a density of 20,000 cells per well were transfected using electroporation with 4,500 nM antisense oligonucleotide. After a treatment period of approximately 24 hours, RNA was isolated from the cells and ANGPTL3 mRNA levels were measured by quantitative real-time PCR. Human primer probe set RTS3492_MGB was used to measure mRNA levels. ANGPTL3 mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of ANGPTL3, relative to untreated control cells.

The newly designed chimeric antisense oligonucleotides in the Tables below were designed as deoxy, MOE, and (S)-cEt oligonucleotides or 5-10-5 MOE gapmers. The deoxy, MOE and (S)-cEt oligonucleotides are 16 nucleosides in length wherein the nucleoside have either a MOE sugar modification, an (S)-cEt sugar modification, or a deoxy sugar residue. The sugar modifications of each antisense oligonucleotide is described as ‘eek-d10-kke’, where ‘k’ indicates an (S)-cEt sugar modification; ‘d’ indicates deoxyribose; the number indicates the number ofdeoxyribose sugars residues; and ‘e’ indicates a MOE modification. The 5-10-5 MOE gapmers are 20 nucleosides in length, wherein the central gap segment comprises often 2′-deoxynucleosides and is flanked by wing segments on the 5′ direction and the 3′ direction comprising five nucleosides each. The internucleoside linkages throughout each oligonucleotide are phosphorothioate (P═S) linkages. All cytosine residues throughout each oligonucleotide are 5-methylcytosines. “Start site” indicates the 5′-most nucleoside to which the oligonucleotide is targeted in the human gene sequence. “Stop site” indicates the 3′-most nucleoside to which the oligonucleotide is targeted human gene sequence. Each oligonucleotide listed in the Tables below is targeted to either the human ANGPTL3 mRNA, designated herein as SEQ ID NO: 1 (GENBANK Accession No. NM_014495.2) or the human ANGPTL3 genomic sequence, designated herein as SEQ ID NO: 2 (GENBANK Accession No. NT_032977.9 truncated from nucleotides 33032001 to 33046000). ‘n/a’ indicates that the antisense oligonucleotide does not target that particular gene sequence with 100% complementarity.

TABLE 150 Inhibition of ANGPTL3 mRNA by oligonucleotides targeting SEQ ID NO: 1 and 2 SEQ SEQ SEQ ID SEQ ID ID ID NO: 1 NO: NO: 2 NO: 2 Start 1 Stop % Start Stop SEQ ID ISIS NO Site Site Sequence Chemistry inhibition Site Site NO 561671 N/A N/A TCTTAACTCTATATAT Deoxy, MOE, and cEt 12 3076 3091 3370 561672 N/A N/A CTTCTTAACTCTATAT Deoxy, MOE, and cEt 12 3078 3093 3371 561673 N/A N/A GACTTCTTAACTCTAT Deoxy, MOE, and cEt 18 3080 3095 3372 561674 N/A N/A TAGACTTCTTAACTCT Deoxy, MOE, and cEt 20 3082 3097 3373 561675 N/A N/A CCTAGACTTCTTAACT Deoxy, MOE, and cEt 9 3084 3099 3374 561676 N/A N/A GACCTAGACTTCTTAA Deoxy, MOE, and cEt 0 3086 3101 3375 561677 N/A N/A CAGACCTAGACTTCTT Deoxy, MOE, and cEt 18 3088 3103 3376 561678 N/A N/A AGCAGACCTAGACTTC Deoxy, MOE, and cEt 26 3090 3105 3377 561679 N/A N/A GAAGCAGACCTAGACT Deoxy, MOE, and cEt 24 3092 3107 3378 561680 N/A N/A TGGAAGCAGACCTAGA Deoxy, MOE, and cEt 30 3094 3109 3379 561758 N/A N/A CTTTAACAAATGGGTT Deoxy, MOE, and cEt 25 11539 11554 3380 561759 N/A N/A ATCCTTTAACAAATGG Deoxy, MOE, and cEt 31 11542 11557 3381 561760 N/A N/A CTATATCCTTTAACAA Deoxy, MOE, and cEt 28 11546 11561 3382 561761 N/A N/A GCACTATATCCTTTAA Deoxy, MOE, and cEt 59 11549 11564 3383 561762 N/A N/A TGGGCACTATATCCTT Deoxy, MOE, and cEt 34 11552 11567 3384 561763 N/A N/A ACTTGGGCACTATATC Deoxy, MOE, and cEt 30 11555 11570 3385 561764 N/A N/A ATAACTTGGGCACTAT Deoxy, MOE, and cEt 51 11558 11573 3386 561765 N/A N/A CATATAACTTGGGCAC Deoxy, MOE, and cEt 47 11561 11576 3387 561766 N/A N/A CACCATATAACTTGGG Deoxy, MOE, and cEt 47 11564 11579 3388 561767 N/A N/A GGTCACCATATAACTT Deoxy, MOE, and cEt 58 11567 11582 3389 561768 N/A N/A GTAGGTCACCATATAA Deoxy, MOE, and cEt 62 11570 11585 3390 561769 N/A N/A AAGGTAGGTCACCATA Deoxy, MOE, and cEt 65 11573 11588 3391 561770 N/A N/A ACAAAGGTAGGTCACC Deoxy, MOE, and cEt 73 11576 11591 143 561771 N/A N/A TTGACAAAGGTAGGTC Deoxy, MOE, and cEt 70 11579 11594 3392 561772 N/A N/A GTATTGACAAAGGTAG Deoxy, MOE, and cEt 58 11582 11597 3393 561773 N/A N/A TAAGTATTGACAAAGG Deoxy, MOE, and cEt 42 11585 11600 3394 561774 N/A N/A TGCTAAGTATTGACAA Deoxy, MOE, and cEt 51 11588 11603 3395 561775 N/A N/A TAATGCTAAGTATTGA Deoxy, MOE, and cEt 42 11591 11606 3396 561776 N/A N/A TACATAATGCTAAGTA Deoxy, MOE, and cEt 36 11595 11610 3397 561777 N/A N/A GGATAATTTGAAATAC Deoxy, MOE, and cEt 24 11608 11623 3398 561778 N/A N/A TATTGGATAATTTGAA Deoxy, MOE, and cEt 35 11612 11627 3399 561779 N/A N/A GTATATTGGATAATTT Deoxy, MOE, and cEt 0 11615 11630 3400 561780 N/A N/A CATGTATATTGGATAA Deoxy, MOE, and cEt 20 11618 11633 3401 561781 N/A N/A TGACATGTATATTGGA Deoxy, MOE, and cEt 73 11621 11636 144 561782 N/A N/A CTTTTATATATGTGAC Deoxy, MOE, and cEt 37 11652 11667 3402 561783 N/A N/A GATCATACATATCTTT Deoxy, MOE, and cEt 51 11664 11679 3403 561784 N/A N/A ATAGATCATACATATC Deoxy, MOE, and cEt 46 11667 11682 3404 561785 N/A N/A CACATAGATCATACAT Deoxy, MOE, and cEt 65 11670 11685 3405 561786 N/A N/A ATTCACATAGATCATA Deoxy, MOE, and cEt 48 11673 11688 3406 561787 N/A N/A AGGATTCACATAGATC Deoxy, MOE, and cEt 48 11676 11691 3407 561788 N/A N/A CTTAGGATTCACATAG Deoxy, MOE, and cEt 42 11679 11694 3408 561789 N/A N/A TTACTTAGGATTCACA Deoxy, MOE, and cEt 58 11682 11697 3409 561790 N/A N/A TATTTACTTAGGATTC Deoxy, MOE, and cEt 45 11685 11700 3410 561791 N/A N/A GTACTTTTCTGGAACA Deoxy, MOE, and cEt 77 11704 11719 145 561792 N/A N/A CCTGAAAATTATAGAT Deoxy, MOE, and cEt 35 11741 11756 3411 561793 N/A N/A GGTCCTGAAAATTATA Deoxy, MOE, and cEt 32 11744 11759 3412 561794 N/A N/A TGTGGTCCTGAAAATT Deoxy, MOE, and cEt 45 11747 11762 3413 561795 N/A N/A GTCTGTGGTCCTGAAA Deoxy, MOE, and cEt 47 11750 11765 3414 561796 N/A N/A TTAGTCTGTGGTCCTG Deoxy, MOE, and cEt 67 11753 11768 3415 561797 N/A N/A AGCTTAGTCTGTGGTC Deoxy, MOE, and cEt 55 11756 11771 3416 561798 N/A N/A GACAGCTTAGTCTGTG Deoxy, MOE, and cEt 47 11759 11774 3417 561799 N/A N/A TTCGACAGCTTAGTCT Deoxy, MOE, and cEt 68 11762 11777 3418 561800 N/A N/A AATTTCGACAGCTTAG Deoxy, MOE, and cEt 61 11765 11780 3419 561801 N/A N/A GTTAATTTCGACAGCT Deoxy, MOE, and cEt 70 11768 11783 3420 561802 N/A N/A CCTAAAAAAATCAGCG Deoxy, MOE, and cEt 19 11783 11798 3421 561803 N/A N/A GGCCCTAAAAAAATCA Deoxy, MOE, and cEt 0 11786 11801 3422 561804 N/A N/A TTCTGGCCCTAAAAAA Deoxy, MOE, and cEt 10 11790 11805 3423 561805 N/A N/A GTATTCTGGCCCTAAA Deoxy, MOE, and cEt 44 11793 11808 3424 561806 N/A N/A TTGGTATTCTGGCCCT Deoxy, MOE, and cEt 45 11796 11811 3425 561807 N/A N/A ATTTTGGTATTCTGGC Deoxy, MOE, and cEt 59 11799 11814 3426 561808 N/A N/A GCCATTTTGGTATTCT Deoxy, MOE, and cEt 58 11802 11817 3427 561809 N/A N/A GGAGCCATTTTGGTAT Deoxy, MOE, and cEt 33 11805 11820 3428 561810 N/A N/A AGAGGAGCCATTTTGG Deoxy, MOE, and cEt 36 11808 11823 3429 561811 N/A N/A AAGAGAGGAGCCATTT Deoxy, MOE, and cEt 14 11811 11826 3430 561812 N/A N/A ATTGTCCAATTTTGGG Deoxy, MOE, and cEt 25 11829 11844 3431 561813 N/A N/A GAAATTGTCCAATTTT Deoxy, MOE, and cEt 38 11832 11847 3432 561814 N/A N/A TTTGAAATTGTCCAAT Deoxy, MOE, and cEt 36 11835 11850 3433 561815 N/A N/A GCATTTGAAATTGTCC Deoxy, MOE, and cEt 67 11838 11853 3434 561816 N/A N/A GCAACTCATATATTAA Deoxy, MOE, and cEt 57 11869 11884 3435 561817 N/A N/A GAAGCAACTCATATAT Deoxy, MOE, and cEt 46 11872 11887 3436 561818 N/A N/A GAGGAAGCAACTCATA Deoxy, MOE, and cEt 14 11875 11890 3437 561819 N/A N/A ATAGAGGAAGCAACTC Deoxy, MOE, and cEt 60 11878 11893 3438 561820 N/A N/A CAAATAGAGGAAGCAA Deoxy, MOE, and cEt 36 11881 11896 3439 561821 N/A N/A AACCAAATAGAGGAAG Deoxy, MOE, and cEt 38 11884 11899 3440 561822 N/A N/A GGAAACCAAATAGAGG Deoxy, MOE, and cEt 51 11887 11902 3441 561823 N/A N/A CTTTAAGTGAAGTTAC Deoxy, MOE, and cEt 30 3636 3651 3442 561824 N/A N/A TACTTACTTTAAGTGA Deoxy, MOE, and cEt 27 3642 3657 3443 561825 N/A N/A GAACCCTCTTTATTTT Deoxy, MOE, and cEt 25 3659 3674 3444 561826 N/A N/A AAACATGAACCCTCTT Deoxy, MOE, and cEt 14 3665 3680 3445 561827 N/A N/A GATCCACATTGAAAAC Deoxy, MOE, and cEt 0 3683 3698 3446 561828 N/A N/A CATGCCTTAGAAATAT Deoxy, MOE, and cEt 33 3710 3725 3447 561829 N/A N/A AAATGGCATGCCTTAG Deoxy, MOE, and cEt 46 3716 3731 3448 561830 N/A N/A GTATTTCAAATGGCAT Deoxy, MOE, and cEt 54 3723 3738 3449 561831 N/A N/A GCAACAAAGTATTTCA Deoxy, MOE, and cEt 60 3731 3746 3450 561832 N/A N/A GTATTTCAACAATGCA Deoxy, MOE, and cEt 28 3744 3759 3451 561833 N/A N/A ATAACATTAGGGAAAC Deoxy, MOE, and cEt 18 3827 3842 3452 561834 N/A N/A TCATATATAACATTAG Deoxy, MOE, and cEt 18 3833 3848 3453 561912 N/A N/A GTGGTTTTGAGCAAAG Deoxy, MOE, and cEt 5 4736 4751 3454 561913 N/A N/A CTATTGTGTGGTTTTG Deoxy, MOE, and cEt 36 4743 4758 3455 561914 N/A N/A GGAAAGCTATTGTGTG Deoxy, MOE, and cEt 18 4749 4764 3456 561915 N/A N/A TATGAGTGAAATGGAA Deoxy, MOE, and cEt 13 4761 4776 3457 561916 N/A N/A AGCCAATATGAGTGAA Deoxy, MOE, and cEt 57 4767 4782 3458 561917 N/A N/A CTAAAGAGCCAATATG Deoxy, MOE, and cEt 33 4773 4788 3459 561918 N/A N/A CTTGGTCTAAAGAGCC Deoxy, MOE, and cEt 70 4779 4794 146 561919 N/A N/A GGTAATCTTGGTCTAA Deoxy, MOE, and cEt 46 4785 4800 3460 561920 N/A N/A GATGACGAAGGGTTGG Deoxy, MOE, and cEt 28 4800 4815 3461 561921 N/A N/A CAGTGAGATGACGAAG Deoxy, MOE, and cEt 39 4806 4821 3462 561922 N/A N/A TGAAGTCAGTGAGATG Deoxy, MOE, and cEt 49 4812 4827 3463 561923 N/A N/A AGGAGGTGAAGTCAGT Deoxy, MOE, and cEt 35 4818 4833 3464 561924 N/A N/A GAGTAGAGGAGGTGAA Deoxy, MOE, and cEt 33 4824 4839 3465 561925 N/A N/A TAACTAGAGTAGAGGA Deoxy, MOE, and cEt 35 4830 4845 3466 561926 N/A N/A TCAGAATAACTAGAGT Deoxy, MOE, and cEt 24 4836 4851 3467 561927 N/A N/A AAGCGGTCAGAATAAC Deoxy, MOE, and cEt 39 4842 4857 3468 561928 N/A N/A CTGGTAAAGCGGTCAG Deoxy, MOE, and cEt 51 4848 4863 3469 561929 N/A N/A TGAATACTGGTAAAGC Deoxy, MOE, and cEt 63 4854 4869 3470 561930 N/A N/A TGTGTTTGAATACTGG Deoxy, MOE, and cEt 65 4860 4875 3471 561931 N/A N/A GTTTGATGTGTTTGAA Deoxy, MOE, and cEt 49 4866 4881 3472 561932 N/A N/A CAGTATGTTTGATGTG Deoxy, MOE, and cEt 48 4872 4887 3473 561933 N/A N/A AGGTGGCAGTATGTTT Deoxy, MOE, and cEt 0 4878 4893 3474 561934 N/A N/A GCTTTGAGGTGGCAGT Deoxy, MOE, and cEt 48 4884 4899 3475 561935 N/A N/A GGGCAAAGGCTTTGAG Deoxy, MOE, and cEt 28 4892 4907 3476 561936 N/A N/A CAACAAGGGCAAAGGC Deoxy, MOE, and cEt 65 4898 4913 3477 561937 N/A N/A GAGGAAACAACAAGGG Deoxy, MOE, and cEt 42 4905 4920 3478 561938 N/A N/A CCAGTTAGAGGAAACA Deoxy, MOE, and cEt 52 4912 4927 3479 561939 N/A N/A CCAGGGCAGAAGAGCG Deoxy, MOE, and cEt 61 4930 4945 3480 561940 N/A N/A TAGATACCAGGGCAGA Deoxy, MOE, and cEt 68 4936 4951 3481 561941 N/A N/A CAGAGAGTGGGCCACG Deoxy, MOE, and cEt 46 4952 4967 3482 561942 N/A N/A GGAAATCAGAGAGTGG Deoxy, MOE, and cEt 42 4958 4973 3483 561943 N/A N/A CCTAAGGGAAATCAGA Deoxy, MOE, and cEt 26 4964 4979 3484 561944 N/A N/A AACGACCCTAAGGGAA Deoxy, MOE, and cEt 45 4970 4985 3485 561945 N/A N/A TTTGATAACGACCCTA Deoxy, MOE, and cEt 57 4976 4991 3486 561946 N/A N/A TTTTTGTTTGATAACG Deoxy, MOE, and cEt 21 4982 4997 3487 561947 N/A N/A CATTGGGAATTTTTTG Deoxy, MOE, and cEt 35 4992 5007 3488 561948 N/A N/A AGTCTTCATTGGGAAT Deoxy, MOE, and cEt 69 4998 5013 3489 561949 N/A N/A CTTGTAAGTCTTCATT Deoxy, MOE, and cEt 35 5004 5019 3490 561950 N/A N/A AGTGACCTTGTAAGTC Deoxy, MOE, and cEt 56 5010 5025 3491 561951 N/A N/A TGGTTAAGTGACCTTG Deoxy, MOE, and cEt 67 5016 5031 3492 561952 N/A N/A GATTTTTGGTTAAGTG Deoxy, MOE, and cEt 43 5022 5037 3493 561953 N/A N/A GGTTGTGATTTTTGGT Deoxy, MOE, and cEt 58 5028 5043 3494 561954 N/A N/A CCAGGCGGTTGTGATT Deoxy, MOE, and cEt 49 5034 5049 3495 561955 N/A N/A ATGGGACCAGGCGGTT Deoxy, MOE, and cEt 52 5040 5055 3496 561956 N/A N/A AAGTTTTCAGGGATGG Deoxy, MOE, and cEt 49 5052 5067 3497 561957 N/A N/A AAGTAGAAGTTTTCAG Deoxy, MOE, and cEt 16 5058 5073 3498 561958 N/A N/A CTAAGGAAGTAGAAGT Deoxy, MOE, and cEt 33 5064 5079 3499 561959 N/A N/A AAGTAGCTAAGGAAGT Deoxy, MOE, and cEt 35 5070 5085 3500 561960 N/A N/A GGAGAAAAGTAGCTAA Deoxy, MOE, and cEt 36 5076 5091 3501 561961 N/A N/A TGTGCAGGAGAAAAGT Deoxy, MOE, and cEt 53 5082 5097 3502 561962 N/A N/A GGTGAGTGTGCAGGAG Deoxy, MOE, and cEt 44 5088 5103 3503 561963 N/A N/A AATAAAGGTGAGTGTG Deoxy, MOE, and cEt 38 5094 5109 3504 561964 N/A N/A TGCAGGAATAGAAGAG Deoxy, MOE, and cEt 58 5138 5153 3505 561965 N/A N/A TTTTAGTGCAGGAATA Deoxy, MOE, and cEt 20 5144 5159 3506 561966 N/A N/A TATTCACAGAGCTTAC Deoxy, MOE, and cEt 63 5161 5176 3507 561967 N/A N/A TCCCTGTATTCACAGA Deoxy, MOE, and cEt 61 5167 5182 3508 561968 N/A N/A GAAAAAATCCCTGTAT Deoxy, MOE, and cEt 22 5174 5189 3509 561969 N/A N/A TATGAAGATAATGGAA Deoxy, MOE, and cEt 34 5187 5202 3510 561970 N/A N/A GGAGTATATACAAATA Deoxy, MOE, and cEt 46 5211 5226 3511 561971 N/A N/A TATTCTGGAGTATATA Deoxy, MOE, and cEt 29 5217 5232 3512 561972 N/A N/A ATTCTATATTCTGGAG Deoxy, MOE, and cEt 58 5223 5238 3513 561973 N/A N/A CATACAGTATTCTATA Deoxy, MOE, and cEt 39 5231 5246 3514 561974 N/A N/A GTGTGCCATACAGTAT Deoxy, MOE, and cEt 48 5237 5252 3515 561975 N/A N/A AGAAATGCCTACTGTG Deoxy, MOE, and cEt 34 5250 5265 3516 561976 N/A N/A ATTCAACAGAAATGCC Deoxy, MOE, and cEt 52 5257 5272 3517 561977 N/A N/A GAATATGACATTACAT Deoxy, MOE, and cEt 33 5279 5294 3518 561978 N/A N/A CTGTGTGAATATGACA Deoxy, MOE, and cEt 63 5285 5300 3519 561979 N/A N/A ACGCTTCTGTGTGAAT Deoxy, MOE, and cEt 59 5291 5306 3520 561980 N/A N/A TAGCACACGCTTCTGT Deoxy, MOE, and cEt 29 5297 5312 3521 561981 N/A N/A TAATCATAGCACACGC Deoxy, MOE, and cEt 64 5303 5318 3522 561982 N/A N/A CCAAGTAATAATAATC Deoxy, MOE, and cEt 26 5314 5329 3523 561983 N/A N/A AGTAATCCAAGTAATA Deoxy, MOE, and cEt 33 5320 5335 3524 561984 N/A N/A ATTTCTAGTAATCCAA Deoxy, MOE, and cEt 42 5326 5341 3525 561985 N/A N/A CACACTATTTCTAGTA Deoxy, MOE, and cEt 40 5332 5347 3526 561986 N/A N/A ATGAGGCACACTATTT Deoxy, MOE, and cEt 47 5338 5353 3527 561987 N/A N/A TTAATTATGAGGCACA Deoxy, MOE, and cEt 58 5344 5359 3528 561988 N/A N/A TGACCTTTAATTATGA Deoxy, MOE, and cEt 38 5350 5365 3529 562066 N/A N/A GCAATTTATTGAATGA Deoxy, MOE, and cEt 27 6083 6098 3530 562067 N/A N/A GGGTTTGCAATTTATT Deoxy, MOE, and cEt 38 6089 6104 3531 562068 N/A N/A TGTGTTGGGTTTGCAA Deoxy, MOE, and cEt 43 6095 6110 3532 562069 N/A N/A TTTAAGTGTGTTGGGT Deoxy, MOE, and cEt 71 6101 6116 3533 562070 N/A N/A GTTTAGCAGTAACATT Deoxy, MOE, and cEt 38 6126 6141 3534 562071 N/A N/A ATTCAGTAGTTTATCG Deoxy, MOE, and cEt 17 6145 6160 3535 562072 N/A N/A CTATATATTCAGTAGT Deoxy, MOE, and cEt 0 6151 6166 3536 562073 N/A N/A GCTTACTTTCTATATA Deoxy, MOE, and cEt 21 6160 6175 3537 562074 N/A N/A AGTTTGTTTGCTTACT Deoxy, MOE, and cEt 63 6169 6184 3538 562075 N/A N/A TTGGCAAGTTTGTTTG Deoxy, MOE, and cEt 55 6175 6190 3539 562076 N/A N/A GGCAGGTTGGCAAGTT Deoxy, MOE, and cEt 68 6181 6196 3540 562077 N/A N/A GATGTTGGCAGGTTGG Deoxy, MOE, and cEt 54 6187 6202 3541 562078 N/A N/A TCTGTAGATGTTGGCA Deoxy, MOE, and cEt 81 6193 6208 147 562079 N/A N/A AACATATCTGTAGATG Deoxy, MOE, and cEt 32 6199 6214 3542 562080 N/A N/A CCTGTAAACATATCTG Deoxy, MOE, and cEt 51 6205 6220 3543 562081 N/A N/A TTTTGACCTGTAAACA Deoxy, MOE, and cEt 14 6211 6226 3544 562082 N/A N/A GATAATTTTTGACCTG Deoxy, MOE, and cEt 49 6217 6232 3545 562083 N/A N/A TCTTGATAATTTGATA Deoxy, MOE, and cEt 13 6229 6244 3546 562084 N/A N/A AGGCTTTCTTGATAAT Deoxy, MOE, and cEt 55 6235 6250 3547 562085 N/A N/A TGAACCAGGCTTTCTT Deoxy, MOE, and cEt 74 6241 6256 3548 562086 N/A N/A ATAATTTGAACCAGGC Deoxy, MOE, and cEt 82 6247 6262 148 562087 N/A N/A GATAAAGACATAATAC Deoxy, MOE, and cEt 21 6263 6278 3549 562088 N/A N/A ACCTGTGATAAAGACA Deoxy, MOE, and cEt 27 6269 6284 3550 562089 N/A N/A CTTCAGACCTGTGATA Deoxy, MOE, and cEt 23 6275 6290 3551 562090 N/A N/A ACTGATCTTCAGACCT Deoxy, MOE, and cEt 48 6281 6296 3552 562091 N/A N/A GGTCTTACTGATCTTC Deoxy, MOE, and cEt 59 6287 6302 3553 562092 N/A N/A GTTTTAGGTCTTACTG Deoxy, MOE, and cEt 21 6293 6308 3554 562093 N/A N/A GTTCAGATTTTAAGTT Deoxy, MOE, and cEt 31 6321 6336 3555 562094 N/A N/A ATATTCTGTTCAGATT Deoxy, MOE, and cEt 36 6328 6343 3556 562095 N/A N/A ATATTGTAATGTATTC Deoxy, MOE, and cEt 52 6372 6387 3557 562096 N/A N/A CTTAGAATATTGTAAT Deoxy, MOE, and cEt 13 6378 6393 3558 562097 N/A N/A GCTTTGCTTAGAATAT Deoxy, MOE, and cEt 47 6384 6399 3559 562098 N/A N/A GAGACTGCTTTGCTTA Deoxy, MOE, and cEt 48 6390 6405 3560 562099 N/A N/A AAAGTAGAGACTGCTT Deoxy, MOE, and cEt 44 6396 6411 3561 562100 N/A N/A AGGCCAAAAGTAGAGA Deoxy, MOE, and cEt 59 6402 6417 3562 562101 N/A N/A TCGGAAAACAGAGCAA Deoxy, MOE, and cEt 63 6417 6432 3563 562102 N/A N/A CATTGGTCGGAAAACA Deoxy, MOE, and cEt 53 6423 6438 3564 562103 N/A N/A AGCAGACATTGGTCGG Deoxy, MOE, and cEt 83 6429 6444 149 562104 N/A N/A AGCAAGGCAAAAAAGC Deoxy, MOE, and cEt 22 6442 6457 3565 562105 N/A N/A GACATTATTTAATAAG Deoxy, MOE, and cEt 21 6470 6485 3566 562106 N/A N/A ATCAGGGACATTATTT Deoxy, MOE, and cEt 34 6476 6491 3567 562107 N/A N/A TATTTAATCAGGGACA Deoxy, MOE, and cEt 47 6482 6497 3568 562108 N/A N/A ATTACCTGTTCTCAAA Deoxy, MOE, and cEt 30 6499 6514 3569 562109 N/A N/A GTACAGATTACCTGTT Deoxy, MOE, and cEt 38 6505 6520 3570 562110 N/A N/A CAGATTGTACAGATTA Deoxy, MOE, and cEt 76 6511 6526 150 562111 N/A N/A GTTATTCAGATTGTAC Deoxy, MOE, and cEt 32 6517 6532 3571 562112 N/A N/A AACAGTGTTATTCAGA Deoxy, MOE, and cEt 58 6523 6538 3572 562113 N/A N/A TAGATAAACAGTGTTA Deoxy, MOE, and cEt 33 6529 6544 3573 562114 N/A N/A TGATATTTAGATAAAC Deoxy, MOE, and cEt 26 6536 6551 3574 562115 N/A N/A GGTGTTTGATATTTAG Deoxy, MOE, and cEt 60 6542 6557 3575 562116 N/A N/A TATAACGGTGTTTGAT Deoxy, MOE, and cEt 42 6548 6563 3576 562117 N/A N/A TAATGTTATAACGGTG Deoxy, MOE, and cEt 62 6554 6569 3577 562118 N/A N/A AGTTCATAATGTTATA Deoxy, MOE, and cEt 21 6560 6575 3578 562119 N/A N/A GTCTTTCAGTTCATAA Deoxy, MOE, and cEt 57 6567 6582 3579 562120 N/A N/A ACAGTTTGTCTTTCAG Deoxy, MOE, and cEt 59 6574 6589 3580 562121 N/A N/A AGAAGTACAGTTTGTC Deoxy, MOE, and cEt 3 6580 6595 3581 562122 N/A N/A GATGTCAGAAGTACAG Deoxy, MOE, and cEt 45 6586 6601 3582 562123 N/A N/A AGTAAGGATGTCAGAA Deoxy, MOE, and cEt 44 6592 6607 3583 562124 N/A N/A AATCTGAGTAAGGATG Deoxy, MOE, and cEt 45 6598 6613 3584 562125 N/A N/A GAATATACAATTAGGG Deoxy, MOE, and cEt 13 6616 6631 3585 562126 N/A N/A TGATACTGAATATACA Deoxy, MOE, and cEt 13 6623 6638 3586 562127 N/A N/A CTGAGCTGATAAAAGA Deoxy, MOE, and cEt 1 6660 6675 3587 562128 N/A N/A ACCATCATGTTTTACA Deoxy, MOE, and cEt 44 6772 6787 3588 562129 N/A N/A TGTCTTACCATCATGT Deoxy, MOE, and cEt 29 6778 6793 3589 562130 N/A N/A CCAAAGTGTCTTACCA Deoxy, MOE, and cEt 42 6784 6799 3590 562131 N/A N/A AACCCACCAAAGTGTC Deoxy, MOE, and cEt 33 6790 6805 3591 562132 N/A N/A GAAGGAAACCCACCAA Deoxy, MOE, and cEt 24 6796 6811 3592 562133 N/A N/A CTTCAAGAAGGAAACC Deoxy, MOE, and cEt 28 6802 6817 3593 562134 N/A N/A TAATAGCTTCAAGAAG Deoxy, MOE, and cEt 1 6808 6823 3594 562135 N/A N/A GGGAATTTGATAATAA Deoxy, MOE, and cEt 0 6821 6836 3595 562136 N/A N/A AGAATAGGGAATTTGA Deoxy, MOE, and cEt 18 6827 6842 3596 562137 N/A N/A GTCCTAAGAATAGGGA Deoxy, MOE, and cEt 9 6833 6848 3597 562138 N/A N/A GAACAAGTCCTAAGAA Deoxy, MOE, and cEt 7 6839 6854 3598 562139 N/A N/A AGTCTAGAACAAGTCC Deoxy, MOE, and cEt 70 6845 6860 3599 562140 N/A N/A TCTTTTAGTCTAGAAC Deoxy, MOE, and cEt 22 6851 6866 3600 562141 N/A N/A TAACTATCTTTTAGTC Deoxy, MOE, and cEt 15 6857 6872 3601 562142 N/A N/A ATCTCTTAACTATCTT Deoxy, MOE, and cEt 35 6863 6878 3602 560991 3 18 AACTGTTTTCTTCTGG Deoxy, MOE, and cEt 37 3107 3122 3603 560992 8 23 CGTGGAACTGTTTTCT Deoxy, MOE, and cEt 74 3112 3127 112 560993 22 37 TCAATTTCAAGCAACG Deoxy, MOE, and cEt 68 3126 3141 3604 560994 51 66 CTTAATTGTGAACATT Deoxy, MOE, and cEt 21 3155 3170 3605 560995 53 68 AGCTTAATTGTGAACA Deoxy, MOE, and cEt 59 3157 3172 3606 560996 55 70 GGAGCTTAATTGTGAA Deoxy, MOE, and cEt 0 3159 3174 3607 560997 57 72 AAGGAGCTTAATTGTG Deoxy, MOE, and cEt 36 3161 3176 3608 560998 59 74 AGAAGGAGCTTAATTG Deoxy, MOE, and cEt 47 3163 3178 3609 560999 61 76 AAAGAAGGAGCTTAAT Deoxy, MOE, and cEt 20 3165 3180 3610 561000 76 91 CTAGAGGAACAATAAA Deoxy, MOE, and cEt 23 3180 3195 3611 561001 79 94 TAACTAGAGGAACAAT Deoxy, MOE, and cEt 19 3183 3198 3612 561002 81 96 AATAACTAGAGGAACA Deoxy, MOE, and cEt 38 3185 3200 3613 561003 84 99 GGAAATAACTAGAGGA Deoxy, MOE, and cEt 48 3188 3203 3614 561004 86 101 GAGGAAATAACTAGAG Deoxy, MOE, and cEt 37 3190 3205 3615 561005 88 103 TGGAGGAAATAACTAG Deoxy, MOE, and cEt 68 3192 3207 3616 561006 90 105 TCTGGAGGAAATAACT Deoxy, MOE, and cEt 49 3194 3209 3617 561007 94 109 CAATTCTGGAGGAAAT Deoxy, MOE, and cEt 43 3198 3213 3618 561008 96 111 ATCAATTCTGGAGGAA Deoxy, MOE, and cEt 73 3200 3215 3619 561009 98 113 TGATCAATTCTGGAGG Deoxy, MOE, and cEt 72 3202 3217 3620 561010 100 115 CTTGATCAATTCTGGA Deoxy, MOE, and cEt 82 3204 3219 113 561011 102 117 GTCTTGATCAATTCTG Deoxy, MOE, and cEt 85 3206 3221 114 561012 104 119 TTGTCTTGATCAATTC Deoxy, MOE, and cEt 64 3208 3223 3621 561013 106 121 AATTGTCTTGATCAAT Deoxy, MOE, and cEt 21 3210 3225 3622 561014 108 123 TGAATTGTCTTGATCA Deoxy, MOE, and cEt 66 3212 3227 3623 561015 110 125 GATGAATTGTCTTGAT Deoxy, MOE, and cEt 51 3214 3229 3624 561016 112 127 ATGATGAATTGTCTTG Deoxy, MOE, and cEt 71 3216 3231 3625 561017 115 130 CAAATGATGAATTGTC Deoxy, MOE, and cEt 36 3219 3234 3626 561018 117 132 ATCAAATGATGAATTG Deoxy, MOE, and cEt 27 3221 3236 3627 561019 125 140 GATAGAGAATCAAATG Deoxy, MOE, and cEt 11 3229 3244 3628 561020 129 144 TGGAGATAGAGAATCA Deoxy, MOE, and cEt 73 3233 3248 3629 561021 131 146 TCTGGAGATAGAGAAT Deoxy, MOE, and cEt 51 3235 3250 3630 561022 135 150 TGGCTCTGGAGATAGA Deoxy, MOE, and cEt 76 3239 3254 115 561023 137 152 TTTGGCTCTGGAGATA Deoxy, MOE, and cEt 73 3241 3256 3631 561024 139 154 ATTTTGGCTCTGGAGA Deoxy, MOE, and cEt 61 3243 3258 3632 561025 141 156 TGATTTTGGCTCTGGA Deoxy, MOE, and cEt 83 3245 3260 116 561026 143 158 CTTGATTTTGGCTCTG Deoxy, MOE, and cEt 83 3247 3262 117 561027 145 160 ATCTTGATTTTGGCTC Deoxy, MOE, and cEt 67 3249 3264 3633 559277 147 162 AAATCTTGATTTTGGC Deoxy, MOE, and cEt 75 3251 3266 110 561028 149 164 GCAAATCTTGATTTTG Deoxy, MOE, and cEt 53 3253 3268 3634 561029 151 166 TAGCAAATCTTGATTT Deoxy, MOE, and cEt 27 3255 3270 3635 561030 153 168 CATAGCAAATCTTGAT Deoxy, MOE, and cEt 63 3257 3272 3636 561031 155 170 AACATAGCAAATCTTG Deoxy, MOE, and cEt 56 3259 3274 3637 561032 157 172 CTAACATAGCAAATCT Deoxy, MOE, and cEt 67 3261 3276 3638 561033 159 174 GTCTAACATAGCAAAT Deoxy, MOE, and cEt 51 3263 3278 3639 561034 174 189 TAAAATTTTTACATCG Deoxy, MOE, and cEt 4 3278 3293 3640 561035 177 192 GGCTAAAATTTTTACA Deoxy, MOE, and cEt 0 3281 3296 3641 561036 182 197 CCATTGGCTAAAATTT Deoxy, MOE, and cEt 3 3286 3301 3642 561037 184 199 GGCCATTGGCTAAAAT Deoxy, MOE, and cEt 16 3288 3303 3643 561038 186 201 GAGGCCATTGGCTAAA Deoxy, MOE, and cEt 42 3290 3305 3644 561039 188 203 AGGAGGCCATTGGCTA Deoxy, MOE, and cEt 61 3292 3307 3645 561040 190 205 GAAGGAGGCCATTGGC Deoxy, MOE, and cEt 35 3294 3309 3646 561041 192 207 CTGAAGGAGGCCATTG Deoxy, MOE, and cEt 37 3296 3311 3647 561042 194 209 AACTGAAGGAGGCCAT Deoxy, MOE, and cEt 22 3298 3313 3648 561043 196 211 CCAACTGAAGGAGGCC Deoxy, MOE, and cEt 33 3300 3315 3649 561044 198 213 TCCCAACTGAAGGAGG Deoxy, MOE, and cEt 19 3302 3317 3650 561045 200 215 TGTCCCAACTGAAGGA Deoxy, MOE, and cEt 33 3304 3319 3651 561046 202 217 CATGTCCCAACTGAAG Deoxy, MOE, and cEt 19 3306 3321 3652 561047 204 219 ACCATGTCCCAACTGA Deoxy, MOE, and cEt 19 3308 3323 3653 561048 206 221 AGACCATGTCCCAACT Deoxy, MOE, and cEt 19 3310 3325 3654 561049 208 223 TAAGACCATGTCCCAA Deoxy, MOE, and cEt 0 3312 3327 3655 561050 210 225 TTTAAGACCATGTCCC Deoxy, MOE, and cEt 5 3314 3329 3656 561051 212 227 TCTTTAAGACCATGTC Deoxy, MOE, and cEt 10 3316 3331 3657 561052 214 229 AGTCTTTAAGACCATG Deoxy, MOE, and cEt 10 3318 3333 3658 561053 216 231 AAAGTCTTTAAGACCA Deoxy, MOE, and cEt 29 3320 3335 3659 561054 218 233 ACAAAGTCTTTAAGAC Deoxy, MOE, and cEt 19 3322 3337 3660 561055 220 235 GGACAAAGTCTTTAAG Deoxy, MOE, and cEt 21 3324 3339 3661 561056 222 237 ATGGACAAAGTCTTTA Deoxy, MOE, and cEt 12 3326 3341 3662 561057 224 239 TTATGGACAAAGTCTT Deoxy, MOE, and cEt 10 3328 3343 3663 561058 226 241 TCTTATGGACAAAGTC Deoxy, MOE, and cEt 9 3330 3345 3664 561059 228 243 CGTCTTATGGACAAAG Deoxy, MOE, and cEt 0 3332 3347 3665 561060 242 257 TTAATTTGGCCCTTCG Deoxy, MOE, and cEt 28 3346 3361 3666 561061 244 259 CATTAATTTGGCCCTT Deoxy, MOE, and cEt 13 3348 3363 3667 561062 246 261 GTCATTAATTTGGCCC Deoxy, MOE, and cEt 63 3350 3365 3668 561063 248 263 ATGTCATTAATTTGGC Deoxy, MOE, and cEt 37 3352 3367 3669 561064 267 282 TATGTTGAGTTTTTGA Deoxy, MOE, and cEt 16 3371 3386 3670 561065 272 287 TCAAATATGTTGAGTT Deoxy, MOE, and cEt 21 3376 3391 3671 561066 274 289 GATCAAATATGTTGAG Deoxy, MOE, and cEt 36 3378 3393 3672 560990 709 724 TTCTTGGTGCTCTTGG Deoxy, MOE, and cEt 73 6722 6737 111 337487 804 823 CACTTGTATGTTCACCTCTG 5-10-5 MOE 76 7389 7408 28 561604 1850 1865 GTACAATTACCAGTCC Deoxy, MOE, and cEt 59 10822 10837 3673 561605 1852 1867 CTGTACAATTACCAGT Deoxy, MOE, and cEt 54 10824 10839 3674 561606 1854 1869 AACTGTACAATTACCA Deoxy, MOE, and cEt 57 10826 10841 3675 561607 1856 1871 AGAACTGTACAATTAC Deoxy, MOE, and cEt 36 10828 10843 3676 561608 1858 1873 TAAGAACTGTACAATT Deoxy, MOE, and cEt 29 10830 10845 3677 561609 1862 1877 CATTTAAGAACTGTAC Deoxy, MOE, and cEt 24 10834 10849 3678 561610 1870 1885 TACTACAACATTTAAG Deoxy, MOE, and cEt 1 10842 10857 3679 561611 1874 1889 TTAATACTACAACATT Deoxy, MOE, and cEt 0 10846 10861 3680 561612 1880 1895 TTGAAATTAATACTAC Deoxy, MOE, and cEt 6 10852 10867 3681 561613 1883 1898 GTTTTGAAATTAATAC Deoxy, MOE, and cEt 34 10855 10870 3682 561614 1892 1907 CGATTTTTAGTTTTGA Deoxy, MOE, and cEt 22 10864 10879 3683 561615 1894 1909 GACGATTTTTAGTTTT Deoxy, MOE, and cEt 29 10866 10881 3684 561616 1896 1911 CTGACGATTTTTAGTT Deoxy, MOE, and cEt 50 10868 10883 3685 561617 1898 1913 TGCTGACGATTTTTAG Deoxy, MOE, and cEt 54 10870 10885 3686 561618 1900 1915 TGTGCTGACGATTTTT Deoxy, MOE, and cEt 70 10872 10887 3687 561619 1902 1917 TCTGTGCTGACGATTT Deoxy, MOE, and cEt 69 10874 10889 3688 561620 1904 1919 ACTCTGTGCTGACGAT Deoxy, MOE, and cEt 78 10876 10891 135 561621 1906 1921 ATACTCTGTGCTGACG Deoxy, MOE, and cEt 87 10878 10893 134 561622 1908 1923 ACATACTCTGTGCTGA Deoxy, MOE, and cEt 80 10880 10895 136 561623 1911 1926 TACACATACTCTGTGC Deoxy, MOE, and cEt 61 10883 10898 3689 561624 1913 1928 TTTACACATACTCTGT Deoxy, MOE, and cEt 68 10885 10900 3690 561625 1917 1932 GATTTTTACACATACT Deoxy, MOE, and cEt 17 10889 10904 3691 561626 1946 1961 GAAGCATCAGTTTAAA Deoxy, MOE, and cEt 27 10918 10933 3692 561627 1948 1963 ATGAAGCATCAGTTTA Deoxy, MOE, and cEt 5 10920 10935 3693 561628 1956 1971 GTAGCAAAATGAAGCA Deoxy, MOE, and cEt 73 10928 10943 137 561629 1958 1973 TTGTAGCAAAATGAAG Deoxy, MOE, and cEt 42 10930 10945 3694 561630 1976 1991 CATTTACTCCAAATTA Deoxy, MOE, and cEt 43 10948 10963 3695 561631 1981 1996 TCAAACATTTACTCCA Deoxy, MOE, and cEt 82 10953 10968 138 561632 2006 2021 CATTAGGTTTCATAAA Deoxy, MOE, and cEt 19 10978 10993 3696 561633 2008 2023 TTCATTAGGTTTCATA Deoxy, MOE, and cEt 15 10980 10995 3697 561634 2010 2025 GCTTCATTAGGTTTCA Deoxy, MOE, and cEt 57 10982 10997 3698 561635 2012 2027 CTGCTTCATTAGGTTT Deoxy, MOE, and cEt 0 10984 10999 3699 561636 2014 2029 TTCTGCTTCATTAGGT Deoxy, MOE, and cEt 65 10986 11001 3700 561637 2016 2031 AATTCTGCTTCATTAG Deoxy, MOE, and cEt 48 10988 11003 3701 561638 2024 2039 CAGTATTTAATTCTGC Deoxy, MOE, and cEt 38 10996 11011 3702 561639 2039 2054 GAACTTATTTTAATAC Deoxy, MOE, and cEt 29 11011 11026 3703 561640 2041 2056 GCGAACTTATTTTAAT Deoxy, MOE, and cEt 38 11013 11028 3704 561641 2043 2058 CAGCGAACTTATTTTA Deoxy, MOE, and cEt 46 11015 11030 3705 561642 2045 2060 GACAGCGAACTTATTT Deoxy, MOE, and cEt 64 11017 11032 3706 561643 2047 2062 AAGACAGCGAACTTAT Deoxy, MOE, and cEt 19 11019 11034 3707 561644 2049 2064 TAAAGACAGCGAACTT Deoxy, MOE, and cEt 76 11021 11036 139 561645 2051 2066 TTTAAAGACAGCGAAC Deoxy, MOE, and cEt 49 11023 11038 3708 561646 2053 2068 TGTTTAAAGACAGCGA Deoxy, MOE, and cEt 81 11025 11040 140 561647 2065 2080 GTCATCTCCATTTGTT Deoxy, MOE, and cEt 60 11037 11052 3709 561648 2067 2082 TAGTCATCTCCATTTG Deoxy, MOE, and cEt 69 11039 11054 3710 561649 2069 2084 AGTAGTCATCTCCATT Deoxy, MOE, and cEt 82 11041 11056 141 561650 2071 2086 TTAGTAGTCATCTCCA Deoxy, MOE, and cEt 79 11043 11058 142 561651 2073 2088 ACTTAGTAGTCATCTC Deoxy, MOE, and cEt 66 11045 11060 3711 561652 2075 2090 TGACTTAGTAGTCATC Deoxy, MOE, and cEt 62 11047 11062 3712 561653 2077 2092 TGTGACTTAGTAGTCA Deoxy, MOE, and cEt 52 11049 11064 3713 561654 2079 2094 AATGTGACTTAGTAGT Deoxy, MOE, and cEt 44 11051 11066 3714 561655 2081 2096 TCAATGTGACTTAGTA Deoxy, MOE, and cEt 65 11053 11068 3715 561656 2083 2098 AGTCAATGTGACTTAG Deoxy, MOE, and cEt 70 11055 11070 3716 561657 2085 2100 AAAGTCAATGTGACTT Deoxy, MOE, and cEt 2 11057 11072 3717 561658 2087 2102 TTAAAGTCAATGTGAC Deoxy, MOE, and cEt 15 11059 11074 3718 561659 2089 2104 TGTTAAAGTCAATGTG Deoxy, MOE, and cEt 27 11061 11076 3719 561660 2091 2106 CATGTTAAAGTCAATG Deoxy, MOE, and cEt 51 11063 11078 3720 561661 2093 2108 CTCATGTTAAAGTCAA Deoxy, MOE, and cEt 53 11065 11080 3721 561662 2095 2110 ACCTCATGTTAAAGTC Deoxy, MOE, and cEt 55 11067 11082 3722 561663 2097 2112 ATACCTCATGTTAAAG Deoxy, MOE, and cEt 25 11069 11084 3723 561664 2099 2114 TGATACCTCATGTTAA Deoxy, MOE, and cEt 0 11071 11086 3724 561665 2101 2116 AGTGATACCTCATGTT Deoxy, MOE, and cEt 38 11073 11088 3725 561666 2103 2118 ATAGTGATACCTCATG Deoxy, MOE, and cEt 61 11075 11090 3726 561667 2105 2120 GTATAGTGATACCTCA Deoxy, MOE, and cEt 63 11077 11092 3727 561668 2107 2122 AGGTATAGTGATACCT Deoxy, MOE, and cEt 27 11079 11094 3728 561669 2109 2124 TAAGGTATAGTGATAC Deoxy, MOE, and cEt 34 11081 11096 3729 561670 2111 2126 AATAAGGTATAGTGAT Deoxy, MOE, and cEt 22 11083 11098 3730

TABLE 151 Inhibition of ANGPTL3 mRNA by oligonucleotides targeting SEQ ID NO: 1 and 2 SEQ SEQ SEQ SEQ ID ID ID ID NO: 1 NO: NO: 2 NO: 2 Start 1 Stop % Start Stop SEQ ID ISIS NO Site Site Sequence Chemistry inhibition Site Site NO 562220 N/A N/A GTAAACTTATTGATAA Deoxy, MOE, and cEt 0 7670 7685 3731 562221 N/A N/A GGCATAGTAAACTTAT Deoxy, MOE, and cEt 22 7676 7691 3732 562222 N/A N/A AATTTTGGCATAGTAA Deoxy, MOE, and cEt 0 7682 7697 3733 562223 N/A N/A GGCAATTAATGAATTT Deoxy, MOE, and cEt 15 7693 7708 3734 562224 N/A N/A GTGAAAGGCAATTAAT Deoxy, MOE, and cEt 7 7699 7714 3735 562225 N/A N/A AGTTAAGTGAAAGGCA Deoxy, MOE, and cEt 0 7705 7720 3736 562226 N/A N/A CCCAAAAGTTAAGTGA Deoxy, MOE, and cEt 27 7711 7726 3737 562227 N/A N/A TATGGTCCCAAAAGTT Deoxy, MOE, and cEt 35 7717 7732 3738 562228 N/A N/A ATTTATTATGGTCCCA Deoxy, MOE, and cEt 67 7723 7738 3739 562229 N/A N/A GTTATGGCAATACATT Deoxy, MOE, and cEt 37 7744 7759 3740 562230 N/A N/A ATTAATGTTATGGCAA Deoxy, MOE, and cEt 33 7750 7765 3741 562231 N/A N/A GTAGTTTATTAATGTT Deoxy, MOE, and cEt 15 7757 7772 3742 562232 N/A N/A TGTAAGGTAGTTTATT Deoxy, MOE, and cEt 23 7763 7778 3743 562233 N/A N/A TGGTTTTGTAAGGTAG Deoxy, MOE, and cEt 43 7769 7784 3744 562234 N/A N/A AATTGGTGGTTTTGTA Deoxy, MOE, and cEt 18 7775 7790 3745 562235 N/A N/A GATTTTAATTGGTGGT Deoxy, MOE, and cEt 21 7781 7796 3746 562236 N/A N/A GATGTAAATAACACTT Deoxy, MOE, and cEt 9 7809 7824 3747 562237 N/A N/A TTGACAGATGTAAATA Deoxy, MOE, and cEt 11 7815 7830 3748 562238 N/A N/A TTTATGTTGACAGATG Deoxy, MOE, and cEt 20 7821 7836 3749 562239 N/A N/A AGTAGATTTATGTTGA Deoxy, MOE, and cEt 9 7827 7842 3750 562240 N/A N/A CCTGAATATAATGAAT Deoxy, MOE, and cEt 29 7859 7874 3751 562241 N/A N/A GGACTACCTGAATATA Deoxy, MOE, and cEt 17 7865 7880 3752 562242 N/A N/A ACCATCAAGCCTCCCA Deoxy, MOE, and cEt 45 7956 7971 3753 562243 N/A N/A CCCCTTACCATCAAGC Deoxy, MOE, and cEt 31 7962 7977 3754 562244 N/A N/A TGTAGTCCCCTTACCA Deoxy, MOE, and cEt 16 7968 7983 3755 562245 N/A N/A ATTGAATGTAGTCCCC Deoxy, MOE, and cEt 19 7974 7989 3756 562246 N/A N/A GATTAGCAAGTGAATG Deoxy, MOE, and cEt 6 7994 8009 3757 562247 N/A N/A TTTGTAGATTAGCAAG Deoxy, MOE, and cEt 24 8000 8015 3758 562248 N/A N/A AAGAGGTTCTCAGTAA Deoxy, MOE, and cEt 28 8019 8034 3759 562249 N/A N/A GTCCATAAGAGGTTCT Deoxy, MOE, and cEt 34 8025 8040 3760 562250 N/A N/A TACCTGGTCCATAAGA Deoxy, MOE, and cEt 10 8031 8046 3761 562251 N/A N/A TCCTAATACCTGGTCC Deoxy, MOE, and cEt 32 8037 8052 3762 562252 N/A N/A TACTTTTCCTAATACC Deoxy, MOE, and cEt 20 8043 8058 3763 562253 N/A N/A CGTTACTACTTTTCCT Deoxy, MOE, and cEt 29 8049 8064 3764 562254 N/A N/A CTGAGACTGCTTCTCG Deoxy, MOE, and cEt 36 8067 8082 3765 562255 N/A N/A TGAAGGCTGAGACTGC Deoxy, MOE, and cEt 40 8073 8088 3766 562256 N/A N/A TAAATTATATGAAGGC Deoxy, MOE, and cEt 9 8082 8097 3767 562257 N/A N/A GTAATTGTTTGATAAT Deoxy, MOE, and cEt 0 8097 8112 3768 562258 N/A N/A TACTAACAAATGTGTA Deoxy, MOE, and cEt 0 8110 8125 3769 562259 N/A N/A GTAATTTACTAACAAA Deoxy, MOE, and cEt 0 8116 8131 3770 562260 N/A N/A ATAAGTGTAATTTACT Deoxy, MOE, and cEt 0 8122 8137 3771 562261 N/A N/A GTTGTAATAAGTGTAA Deoxy, MOE, and cEt 0 8128 8143 3772 562262 N/A N/A GTGATAAATATAATTC Deoxy, MOE, and cEt 0 8155 8170 3773 562263 N/A N/A CATGTAATTGTGATAA Deoxy, MOE, and cEt 20 8164 8179 3774 562264 N/A N/A GTATATTTAAGAACAG Deoxy, MOE, and cEt 13 8181 8196 3775 562265 N/A N/A TTGTGATAAGTATATT Deoxy, MOE, and cEt 3 8190 8205 3776 562266 N/A N/A TGGAATTAAATTGTGA Deoxy, MOE, and cEt 0 8200 8215 3777 562267 N/A N/A AAGCCGTGGAATTAAA Deoxy, MOE, and cEt 10 8206 8221 3778 562268 N/A N/A CATTGTAAGCCGTGGA Deoxy, MOE, and cEt 54 8212 8227 3779 562269 N/A N/A TATGATCATTGTAAGC Deoxy, MOE, and cEt 0 8218 8233 3780 562270 N/A N/A TATAGTTATGATCATT Deoxy, MOE, and cEt 0 8224 8239 3781 562271 N/A N/A GACATAACATTTAATC Deoxy, MOE, and cEt 21 8258 8273 3782 562272 N/A N/A ACTTATGACATAACAT Deoxy, MOE, and cEt 14 8264 8279 3783 562273 N/A N/A GTTACTACTTATGACA Deoxy, MOE, and cEt 30 8270 8285 3784 562274 N/A N/A GTAACAGTTACTACTT Deoxy, MOE, and cEt 24 8276 8291 3785 562275 N/A N/A GCTTATTTGTAACAGT Deoxy, MOE, and cEt 17 8284 8299 3786 562276 N/A N/A TTCACAGCTTATTTGT Deoxy, MOE, and cEt 20 8290 8305 3787 562277 N/A N/A GTTCTTTTCACAGCTT Deoxy, MOE, and cEt 46 8296 8311 3788 562278 N/A N/A GGAGTGGTTCTTTTCA Deoxy, MOE, and cEt 35 8302 8317 3789 562279 N/A N/A ATGCTAGGAGTGGTTC Deoxy, MOE, and cEt 29 8308 8323 3790 562280 N/A N/A TGACTAATGCTAGGAG Deoxy, MOE, and cEt 4 8314 8329 3791 562281 N/A N/A ATAGAGTGACTAATGC Deoxy, MOE, and cEt 23 8320 8335 3792 562282 N/A N/A GAGAGAATAGAGTGAC Deoxy, MOE, and cEt 15 8326 8341 3793 562284 N/A N/A ATTGATATGTAAAACG Deoxy, MOE, and cEt 7 8347 8362 3794 562285 N/A N/A CAATTAATTGATATGT Deoxy, MOE, and cEt 14 8353 8368 3795 562286 N/A N/A CCTTTTAACTTCCAAT Deoxy, MOE, and cEt 40 8365 8380 3796 562287 N/A N/A CCTGGTCCTTTTAACT Deoxy, MOE, and cEt 29 8371 8386 3797 562288 N/A N/A GAGTTTCCTGGTCCTT Deoxy, MOE, and cEt 49 8377 8392 3798 562289 N/A N/A ATGTCTGAGTTTCCTG Deoxy, MOE, and cEt 16 8383 8398 3799 562290 N/A N/A TACTGTATGTCTGAGT Deoxy, MOE, and cEt 33 8389 8404 3800 562291 N/A N/A CCATACATTCTATATA Deoxy, MOE, and cEt 10 8437 8452 3801 562292 N/A N/A TATAAGCCATACATTC Deoxy, MOE, and cEt 24 8443 8458 3802 562293 N/A N/A ATTCATTATAAGCCAT Deoxy, MOE, and cEt 38 8449 8464 3803 562295 N/A N/A CATTGAGTTAACTAAT Deoxy, MOE, and cEt 7 8463 8478 3804 562296 N/A N/A AATTTGCATTGAGTTA Deoxy, MOE, and cEt 18 8469 8484 3805 561144 525 540 TGAAGTTACTTCTGGG Deoxy, MOE, and cEt 39 3629 3644 3806 561145 527 542 AGTGAAGTTACTTCTG Deoxy, MOE, and cEt 51 3631 3646 3807 561146 529 544 TAAGTGAAGTTACTTC Deoxy, MOE, and cEt 40 3633 3648 3808 561147 533 548 GTTTTAAGTGAAGTTA Deoxy, MOE, and cEt 29 N/A N/A 3809 561148 535 550 AAGTTTTAAGTGAAGT Deoxy, MOE, and cEt 19 N/A N/A 3810 561149 547 562 GTTTTTCTACAAAAGT Deoxy, MOE, and cEt 38 4285 4300 3811 561150 560 575 ATGCTATTATCTTGTT Deoxy, MOE, and cEt 30 4298 4313 3812 561151 562 577 TGATGCTATTATCTTG Deoxy, MOE, and cEt 36 4300 4315 3813 561152 564 579 TTTGATGCTATTATCT Deoxy, MOE, and cEt 23 4302 4317 3814 561153 567 582 GTCTTTGATGCTATTA Deoxy, MOE, and cEt 51 4305 4320 3815 561154 569 584 AGGTCTTTGATGCTAT Deoxy, MOE, and cEt 60 4307 4322 3816 561155 571 586 GAAGGTCTTTGATGCT Deoxy, MOE, and cEt 61 4309 4324 3817 561156 573 588 GAGAAGGTCTTTGATG Deoxy, MOE, and cEt 30 4311 4326 3818 561157 575 590 TGGAGAAGGTCTTTGA Deoxy, MOE, and cEt 40 4313 4328 3819 561158 577 592 TCTGGAGAAGGTCTTT Deoxy, MOE, and cEt 46 4315 4330 3820 561159 579 594 GGTCTGGAGAAGGTCT Deoxy, MOE, and cEt 57 4317 4332 3821 561160 581 596 ACGGTCTGGAGAAGGT Deoxy, MOE, and cEt 57 4319 4334 3822 561161 583 598 CCACGGTCTGGAGAAG Deoxy, MOE, and cEt 56 4321 4336 3823 561162 585 600 TTCCACGGTCTGGAGA Deoxy, MOE, and cEt 50 4323 4338 3824 561163 587 602 TCTTCCACGGTCTGGA Deoxy, MOE, and cEt 77 4325 4340 3825 561164 589 604 GGTCTTCCACGGTCTG Deoxy, MOE, and cEt 89 4327 4342 3826 561165 591 606 TTGGTCTTCCACGGTC Deoxy, MOE, and cEt 79 4329 4344 3827 561166 593 608 TATTGGTCTTCCACGG Deoxy, MOE, and cEt 39 4331 4346 3828 561167 595 610 TATATTGGTCTTCCAC Deoxy, MOE, and cEt 22 4333 4348 3829 561168 597 612 TTTATATTGGTCTTCC Deoxy, MOE, and cEt 43 4335 4350 3830 561169 599 614 TGTTTATATTGGTCTT Deoxy, MOE, and cEt 50 4337 4352 3831 561170 601 616 ATTGTTTATATTGGTC Deoxy, MOE, and cEt 27 4339 4354 3832 561171 603 618 TAATTGTTTATATTGG Deoxy, MOE, and cEt 21 4341 4356 3833 561172 607 622 GGTTTAATTGTTTATA Deoxy, MOE, and cEt 22 4345 4360 3834 561173 610 625 GTTGGTTTAATTGTTT Deoxy, MOE, and cEt 33 4348 4363 3835 561174 612 627 CTGTTGGTTTAATTGT Deoxy, MOE, and cEt 13 4350 4365 3836 561175 614 629 TGCTGTTGGTTTAATT Deoxy, MOE, and cEt 26 4352 4367 3837 561176 616 631 TATGCTGTTGGTTTAA Deoxy, MOE, and cEt 40 4354 4369 3838 561177 618 633 ACTATGCTGTTGGTTT Deoxy, MOE, and cEt 68 4356 4371 3839 561178 620 635 TGACTATGCTGTTGGT Deoxy, MOE, and cEt 64 4358 4373 3840 561179 622 637 TTTGACTATGCTGTTG Deoxy, MOE, and cEt 42 4360 4375 3841 561180 624 639 TATTTGACTATGCTGT Deoxy, MOE, and cEt 16 4362 4377 3842 561181 626 641 TTTATTTGACTATGCT Deoxy, MOE, and cEt 17 4364 4379 3843 561182 628 643 CTTTTATTTGACTATG Deoxy, MOE, and cEt 7 4366 4381 3844 561183 645 660 GAGCTGATTTTCTATT Deoxy, MOE, and cEt 18 N/A N/A 3845 561184 647 662 CTGAGCTGATTTTCTA Deoxy, MOE, and cEt 42 N/A N/A 3846 561185 649 664 TTCTGAGCTGATTTTC Deoxy, MOE, and cEt 32 N/A N/A 3847 561186 651 666 CCTTCTGAGCTGATTT Deoxy, MOE, and cEt 14 N/A N/A 3848 561187 653 668 GTCCTTCTGAGCTGAT Deoxy, MOE, and cEt 39 6666 6681 3849 561188 655 670 TAGTCCTTCTGAGCTG Deoxy, MOE, and cEt 7 6668 6683 3850 561189 657 672 ACTAGTCCTTCTGAGC Deoxy, MOE, and cEt 32 6670 6685 3851 561190 659 674 ATACTAGTCCTTCTGA Deoxy, MOE, and cEt 19 6672 6687 3852 561191 661 676 GAATACTAGTCCTTCT Deoxy, MOE, and cEt 37 6674 6689 3853 561192 663 678 TTGAATACTAGTCCTT Deoxy, MOE, and cEt 50 6676 6691 3854 561193 665 680 TCTTGAATACTAGTCC Deoxy, MOE, and cEt 28 6678 6693 3855 561194 667 682 GTTCTTGAATACTAGT Deoxy, MOE, and cEt 34 6680 6695 3856 561195 669 684 GGGTTCTTGAATACTA Deoxy, MOE, and cEt 61 6682 6697 3857 561196 671 686 GTGGGTTCTTGAATAC Deoxy, MOE, and cEt 21 6684 6699 3858 561197 673 688 CTGTGGGTTCTTGAAT Deoxy, MOE, and cEt 45 6686 6701 3859 561198 675 690 TTCTGTGGGTTCTTGA Deoxy, MOE, and cEt 0 6688 6703 3860 561199 679 694 AAATTTCTGTGGGTTC Deoxy, MOE, and cEt 31 6692 6707 3861 561200 681 696 AGAAATTTCTGTGGGT Deoxy, MOE, and cEt 60 6694 6709 3862 561201 684 699 TAGAGAAATTTCTGTG Deoxy, MOE, and cEt 35 6697 6712 3863 561202 686 701 GATAGAGAAATTTCTG Deoxy, MOE, and cEt 36 6699 6714 3864 561203 694 709 GCTTGGAAGATAGAGA Deoxy, MOE, and cEt 39 6707 6722 3865 561204 696 711 TGGCTTGGAAGATAGA Deoxy, MOE, and cEt 32 6709 6724 3866 561205 698 713 CTTGGCTTGGAAGATA Deoxy, MOE, and cEt 23 6711 6726 3867 561206 700 715 CTCTTGGCTTGGAAGA Deoxy, MOE, and cEt 21 6713 6728 3868 561207 702 717 TGCTCTTGGCTTGGAA Deoxy, MOE, and cEt 34 6715 6730 3869 561208 704 719 GGTGCTCTTGGCTTGG Deoxy, MOE, and cEt 71 6717 6732 118 561209 706 721 TTGGTGCTCTTGGCTT Deoxy, MOE, and cEt 59 6719 6734 3870 561210 708 723 TCTTGGTGCTCTTGGC Deoxy, MOE, and cEt 65 6721 6736 3871 560990 709 724 TTCTTGGTGCTCTTGG Deoxy, MOE, and cEt 54 6722 6737 111 561211 710 725 GTTCTTGGTGCTCTTG Deoxy, MOE, and cEt 60 6723 6738 3872 561212 712 727 TAGTTCTTGGTGCTCT Deoxy, MOE, and cEt 53 6725 6740 3873 561213 714 729 AGTAGTTCTTGGTGCT Deoxy, MOE, and cEt 50 6727 6742 3874 561214 716 731 GGAGTAGTTCTTGGTG Deoxy, MOE, and cEt 31 6729 6744 3875 561215 718 733 AGGGAGTAGTTCTTGG Deoxy, MOE, and cEt 0 6731 6746 3876 561216 720 735 AAAGGGAGTAGTTCTT Deoxy, MOE, and cEt 25 6733 6748 3877 561217 722 737 AGAAAGGGAGTAGTTC Deoxy, MOE, and cEt 28 6735 6750 3878 561218 724 739 GAAGAAAGGGAGTAGT Deoxy, MOE, and cEt 10 6737 6752 3879 561219 726 741 CTGAAGAAAGGGAGTA Deoxy, MOE, and cEt 47 6739 6754 3880 561220 730 745 TCAACTGAAGAAAGGG Deoxy, MOE, and cEt 50 6743 6758 3881 337487 804 823 CACTTGTATGTTCACCTCTG 5-10-5 MOE 52 7389 7408 28 561297 926 941 TCATTGAAGTTTTGTG Deoxy, MOE, and cEt 28 7913 7928 3882 561298 930 945 CGTTTCATTGAAGTTT Deoxy, MOE, and cEt 35 7917 7932 3883 561299 944 959 TTGTAGTTCTCCCACG Deoxy, MOE, and cEt 30 7931 7946 3884 561300 946 961 ATTTGTAGTTCTCCCA Deoxy, MOE, and cEt 32 7933 7948 3885 561301 948 963 ATATTTGTAGTTCTCC Deoxy, MOE, and cEt 24 7935 7950 3886 561302 950 965 CCATATTTGTAGTTCT Deoxy, MOE, and cEt 5 7937 7952 3887 561303 952 967 AACCATATTTGTAGTT Deoxy, MOE, and cEt 3 7939 7954 3888 561304 956 971 CCAAAACCATATTTGT Deoxy, MOE, and cEt 19 7943 7958 3889 561305 959 974 CTCCCAAAACCATATT Deoxy, MOE, and cEt 23 7946 7961 3890 561306 961 976 GCCTCCCAAAACCATA Deoxy, MOE, and cEt 25 7948 7963 3891 561307 963 978 AAGCCTCCCAAAACCA Deoxy, MOE, and cEt 30 7950 7965 3892 561308 965 980 TCAAGCCTCCCAAAAC Deoxy, MOE, and cEt 16 7952 7967 3893 561309 969 984 TCCATCAAGCCTCCCA Deoxy, MOE, and cEt 46 N/A N/A 3894 561310 971 986 TCTCCATCAAGCCTCC Deoxy, MOE, and cEt 13 N/A N/A 3895 561311 973 988 ATTCTCCATCAAGCCT Deoxy, MOE, and cEt 16 N/A N/A 3896 561312 975 990 AAATTCTCCATCAAGC Deoxy, MOE, and cEt 20 N/A N/A 3897 561313 979 994 ACCAAAATTCTCCATC Deoxy, MOE, and cEt 18 N/A N/A 3898 561314 981 996 CAACCAAAATTCTCCA Deoxy, MOE, and cEt 26 N/A N/A 3899 561315 983 998 CCCAACCAAAATTCTC Deoxy, MOE, and cEt 38 9558 9573 3900 559316 985 1000 GGCCCAACCAAAATTC Deoxy, MOE, and cEt 14 9560 9575 3901 561316 987 1002 TAGGCCCAACCAAAAT Deoxy, MOE, and cEt 38 9562 9577 3902 561317 989 1004 TCTAGGCCCAACCAAA Deoxy, MOE, and cEt 51 9564 9579 3903 561318 991 1006 TCTCTAGGCCCAACCA Deoxy, MOE, and cEt 35 9566 9581 3904 561319 993 1008 CTTCTCTAGGCCCAAC Deoxy, MOE, and cEt 31 9568 9583 3905 561320 995 1010 ATCTTCTCTAGGCCCA Deoxy, MOE, and cEt 68 9570 9585 119 561321 997 1012 ATATCTTCTCTAGGCC Deoxy, MOE, and cEt 30 9572 9587 3906 561322 999 1014 GTATATCTTCTCTAGG Deoxy, MOE, and cEt 25 9574 9589 3907 561323 1001 1016 GAGTATATCTTCTCTA Deoxy, MOE, and cEt 26 9576 9591 3908 561324 1003 1018 TGGAGTATATCTTCTC Deoxy, MOE, and cEt 46 9578 9593 3909 561325 1005 1020 TATGGAGTATATCTTC Deoxy, MOE, and cEt 20 9580 9595 3910 561326 1007 1022 ACTATGGAGTATATCT Deoxy, MOE, and cEt 20 9582 9597 3911 561327 1009 1024 TCACTATGGAGTATAT Deoxy, MOE, and cEt 22 9584 9599 3912 561328 1011 1026 CTTCACTATGGAGTAT Deoxy, MOE, and cEt 33 9586 9601 3913 561329 1013 1028 TGCTTCACTATGGAGT Deoxy, MOE, and cEt 50 9588 9603 3914 561330 1015 1030 ATTGCTTCACTATGGA Deoxy, MOE, and cEt 43 9590 9605 3915 561331 1017 1032 AGATTGCTTCACTATG Deoxy, MOE, and cEt 31 9592 9607 3916 561332 1019 1034 TTAGATTGCTTCACTA Deoxy, MOE, and cEt 36 9594 9609 3917 561333 1021 1036 AATTAGATTGCTTCAC Deoxy, MOE, and cEt 17 9596 9611 3918 561334 1023 1038 ATAATTAGATTGCTTC Deoxy, MOE, and cEt 23 9598 9613 3919 561335 1025 1040 ACATAATTAGATTGCT Deoxy, MOE, and cEt 13 9600 9615 3920 561336 1031 1046 CGTAAAACATAATTAG Deoxy, MOE, and cEt 25 9606 9621 3921 561337 1045 1060 CTTCCAACTCAATTCG Deoxy, MOE, and cEt 0 9620 9635 3922 561338 1047 1062 GTCTTCCAACTCAATT Deoxy, MOE, and cEt 0 9622 9637 3923 561339 1049 1064 CAGTCTTCCAACTCAA Deoxy, MOE, and cEt 15 9624 9639 3924 561340 1051 1066 TCCAGTCTTCCAACTC Deoxy, MOE, and cEt 22 9626 9641 3925 561341 1053 1068 TTTCCAGTCTTCCAAC Deoxy, MOE, and cEt 2 9628 9643 3926 561342 1056 1071 GTCTTTCCAGTCTTCC Deoxy, MOE, and cEt 45 9631 9646 3927 561343 1059 1074 GTTGTCTTTCCAGTCT Deoxy, MOE, and cEt 67 9634 9649 120 561344 1061 1076 TTGTTGTCTTTCCAGT Deoxy, MOE, and cEt 43 9636 9651 3928 561345 1063 1078 GTTTGTTGTCTTTCCA Deoxy, MOE, and cEt 57 9638 9653 121 561346 1068 1083 ATAATGTTTGTTGTCT Deoxy, MOE, and cEt 6 9643 9658 3929 561347 1098 1113 GTGATTTCCCAAGTAA Deoxy, MOE, and cEt 66 9673 9688 122 561348 1113 1128 CGTATAGTTGGTTTCG Deoxy, MOE, and cEt 54 9688 9703 3930 561349 1127 1142 GCAACTAGATGTAGCG Deoxy, MOE, and cEt 50 9702 9717 3931 561350 1129 1144 TCGCAACTAGATGTAG Deoxy, MOE, and cEt 9 9704 9719 3932 561351 1131 1146 AATCGCAACTAGATGT Deoxy, MOE, and cEt 9 9706 9721 3933 561352 1133 1148 GTAATCGCAACTAGAT Deoxy, MOE, and cEt 15 9708 9723 3934 561353 1135 1150 CAGTAATCGCAACTAG Deoxy, MOE, and cEt 41 9710 9725 3935 561354 1137 1152 GCCAGTAATCGCAACT Deoxy, MOE, and cEt 38 9712 9727 3936 561355 1139 1154 TTGCCAGTAATCGCAA Deoxy, MOE, and cEt 32 9714 9729 3937 561356 1141 1156 CATTGCCAGTAATCGC Deoxy, MOE, and cEt 54 9716 9731 3938 561357 1143 1158 GACATTGCCAGTAATC Deoxy, MOE, and cEt 20 9718 9733 3939 561358 1145 1160 GGGACATTGCCAGTAA Deoxy, MOE, and cEt 0 9720 9735 3940 561359 1160 1175 TCCGGGATTGCATTGG Deoxy, MOE, and cEt 43 9735 9750 3941 561360 1162 1177 TTTCCGGGATTGCATT Deoxy, MOE, and cEt 31 9737 9752 3942 561361 1164 1179 GTTTTCCGGGATTGCA Deoxy, MOE, and cEt 31 9739 9754 3943 561362 1166 1181 TTGTTTTCCGGGATTG Deoxy, MOE, and cEt 36 9741 9756 3944 561363 1168 1183 CTTTGTTTTCCGGGAT Deoxy, MOE, and cEt 22 9743 9758 3945 561364 1170 1185 ATCTTTGTTTTCCGGG Deoxy, MOE, and cEt 13 9745 9760 3946 561365 1172 1187 AAATCTTTGTTTTCCG Deoxy, MOE, and cEt 7 9747 9762 3947 561366 1177 1192 ACACCAAATCTTTGTT Deoxy, MOE, and cEt 8 9752 9767 3948 561367 1179 1194 AAACACCAAATCTTTG Deoxy, MOE, and cEt 11 9754 9769 3949 561368 1187 1202 CAAGTAGAAAACACCA Deoxy, MOE, and cEt 16 9762 9777 3950 561369 1189 1204 CCCAAGTAGAAAACAC Deoxy, MOE, and cEt 23 9764 9779 3951 561370 1191 1206 ATCCCAAGTAGAAAAC Deoxy, MOE, and cEt 27 9766 9781 3952 561371 1193 1208 TGATCCCAAGTAGAAA Deoxy, MOE, and cEt 25 9768 9783 3953 561372 1195 1210 TGTGATCCCAAGTAGA Deoxy, MOE, and cEt 45 9770 9785 3954

TABLE 152 Inhibition of ANGPTL3 mRNA by oligonucleotides targeting SEQ ID NO: 1 and 2 SEQ ID SEQ ID SEQ ID SEQ ID NO: 1 NO: NO: 2 NO: 2 Start 1 Stop % Start Stop SEQ ID ISIS NO Site Site Sequence Chemistry inhibition Site Site NO 561067 276 291 CTGATCAAATATGTTG Deoxy, MOE, and cEt 54 3380 3395 3955 561068 278 293 GACTGATCAAATATGT Deoxy, MOE, and cEt 19 3382 3397 3956 561069 280 295 AAGACTGATCAAATAT Deoxy, MOE, and cEt 17 3384 3399 3957 561070 286 301 CATAAAAAGACTGATC Deoxy, MOE, and cEt 18 3390 3405 3958 561071 289 304 GATCATAAAAAGACTG Deoxy, MOE, and cEt 11 3393 3408 3959 561072 291 306 TAGATCATAAAAAGAC Deoxy, MOE, and cEt 0 3395 3410 3960 561073 293 308 GATAGATCATAAAAAG Deoxy, MOE, and cEt 15 3397 3412 3961 561074 295 310 GCGATAGATCATAAAA Deoxy, MOE, and cEt 39 3399 3414 3962 561075 297 312 CAGCGATAGATCATAA Deoxy, MOE, and cEt 53 3401 3416 3963 561076 299 314 TGCAGCGATAGATCAT Deoxy, MOE, and cEt 70 3403 3418 159 561077 301 316 TTTGCAGCGATAGATC Deoxy, MOE, and cEt 60 3405 3420 3964 561078 303 318 GGTTTGCAGCGATAGA Deoxy, MOE, and cEt 63 3407 3422 3965 561079 305 320 CTGGTTTGCAGCGATA Deoxy, MOE, and cEt 76 3409 3424 160 561080 307 322 CACTGGTTTGCAGCGA Deoxy, MOE, and cEt 65 3411 3426 3966 561081 309 324 TTCACTGGTTTGCAGC Deoxy, MOE, and cEt 45 3413 3428 3967 561082 311 326 ATTTCACTGGTTTGCA Deoxy, MOE, and cEt 56 3415 3430 3968 561083 313 328 TGATTTCACTGGTTTG Deoxy, MOE, and cEt 65 3417 3432 3969 561084 316 331 CTTTGATTTCACTGGT Deoxy, MOE, and cEt 73 3420 3435 161 561085 341 356 GTTCTTCTCAGTTCCT Deoxy, MOE, and cEt 79 3445 3460 162 561086 343 358 TAGTTCTTCTCAGTTC Deoxy, MOE, and cEt 50 3447 3462 3970 561087 345 360 TGTAGTTCTTCTCAGT Deoxy, MOE, and cEt 42 3449 3464 3971 561088 347 362 TATGTAGTTCTTCTCA Deoxy, MOE, and cEt 27 3451 3466 3972 561089 349 364 TATATGTAGTTCTTCT Deoxy, MOE, and cEt 37 3453 3468 3973 561090 352 367 GTTTATATGTAGTTCT Deoxy, MOE, and cEt 39 3456 3471 3974 561091 355 370 GTAGTTTATATGTAGT Deoxy, MOE, and cEt 55 3459 3474 3975 561092 358 373 CTTGTAGTTTATATGT Deoxy, MOE, and cEt 48 3462 3477 3976 561093 360 375 GACTTGTAGTTTATAT Deoxy, MOE, and cEt 43 3464 3479 3977 561094 362 377 TTGACTTGTAGTTTAT Deoxy, MOE, and cEt 35 3466 3481 3978 561095 365 380 TTTTTGACTTGTAGTT Deoxy, MOE, and cEt 37 3469 3484 3979 561096 367 382 CATTTTTGACTTGTAG Deoxy, MOE, and cEt 34 3471 3486 3980 561097 373 388 CCTCTTCATTTTTGAC Deoxy, MOE, and cEt 48 3477 3492 3981 561098 386 401 GACATATTCTTTACCT Deoxy, MOE, and cEt 40 3490 3505 3982 561099 388 403 GTGACATATTCTTTAC Deoxy, MOE, and cEt 43 3492 3507 3983 561100 393 408 TTCAAGTGACATATTC Deoxy, MOE, and cEt 51 3497 3512 3984 561101 395 410 AGTTCAAGTGACATAT Deoxy, MOE, and cEt 27 3499 3514 3985 561102 397 412 TGAGTTCAAGTGACAT Deoxy, MOE, and cEt 63 3501 3516 3986 561103 399 414 GTTGAGTTCAAGTGAC Deoxy, MOE, and cEt 48 3503 3518 3987 561104 401 416 GAGTTGAGTTCAAGTG Deoxy, MOE, and cEt 57 3505 3520 3988 561105 403 418 TTGAGTTGAGTTCAAG Deoxy, MOE, and cEt 32 3507 3522 3989 561106 405 420 TTTTGAGTTGAGTTCA Deoxy, MOE, and cEt 47 3509 3524 3990 561107 407 422 AGTTTTGAGTTGAGTT Deoxy, MOE, and cEt 46 3511 3526 3991 561108 409 424 CAAGTTTTGAGTTGAG Deoxy, MOE, and cEt 48 3513 3528 3992 561109 411 426 TTCAAGTTTTGAGTTG Deoxy, MOE, and cEt 17 3515 3530 3993 561110 413 428 CTTTCAAGTTTTGAGT Deoxy, MOE, and cEt 48 3517 3532 3994 561111 415 430 GGCTTTCAAGTTTTGA Deoxy, MOE, and cEt 56 3519 3534 3995 561112 417 432 GAGGCTTTCAAGTTTT Deoxy, MOE, and cEt 39 3521 3536 3996 561113 419 434 AGGAGGCTTTCAAGTT Deoxy, MOE, and cEt 49 3523 3538 3997 561114 421 436 CTAGGAGGCTTTCAAG Deoxy, MOE, and cEt 49 3525 3540 3998 561115 423 438 TTCTAGGAGGCTTTCA Deoxy, MOE, and cEt 40 3527 3542 3999 561116 425 440 TCTTCTAGGAGGCTTT Deoxy, MOE, and cEt 66 3529 3544 4000 561117 427 442 TTTCTTCTAGGAGGCT Deoxy, MOE, and cEt 74 3531 3546 4001 561118 442 457 GTTGAAGTAGAATTTT Deoxy, MOE, and cEt 40 3546 3561 4002 561119 469 484 GTTGCTCTTCTAAATA Deoxy, MOE, and cEt 44 3573 3588 4003 561120 471 486 TAGTTGCTCTTCTAAA Deoxy, MOE, and cEt 19 3575 3590 4004 561121 473 488 GTTAGTTGCTCTTCTA Deoxy, MOE, and cEt 67 3577 3592 4005 561122 475 490 TAGTTAGTTGCTCTTC Deoxy, MOE, and cEt 51 3579 3594 4006 561123 477 492 GTTAGTTAGTTGCTCT Deoxy, MOE, and cEt 73 3581 3596 163 561124 479 494 AAGTTAGTTAGTTGCT Deoxy, MOE, and cEt 51 3583 3598 4007 561125 481 496 TTAAGTTAGTTAGTTG Deoxy, MOE, and cEt 33 3585 3600 4008 561126 483 498 AATTAAGTTAGTTAGT Deoxy, MOE, and cEt 0 3587 3602 4009 561127 485 500 TGAATTAAGTTAGTTA Deoxy, MOE, and cEt 5 3589 3604 4010 561128 487 502 TTTGAATTAAGTTAGT Deoxy, MOE, and cEt 18 3591 3606 4011 561129 494 509 GGTTGATTTTGAATTA Deoxy, MOE, and cEt 20 3598 3613 4012 561130 496 511 CAGGTTGATTTTGAAT Deoxy, MOE, and cEt 27 3600 3615 4013 561131 498 513 TTCAGGTTGATTTTGA Deoxy, MOE, and cEt 33 3602 3617 4014 561132 500 515 GTTTCAGGTTGATTTT Deoxy, MOE, and cEt 38 3604 3619 4015 561133 502 517 GAGTTTCAGGTTGATT Deoxy, MOE, and cEt 33 3606 3621 4016 561134 504 519 TGGAGTTTCAGGTTGA Deoxy, MOE, and cEt 67 3608 3623 4017 561135 507 522 TTCTGGAGTTTCAGGT Deoxy, MOE, and cEt 32 3611 3626 4018 561136 509 524 TGTTCTGGAGTTTCAG Deoxy, MOE, and cEt 14 3613 3628 4019 561137 511 526 GGTGTTCTGGAGTTTC Deoxy, MOE, and cEt 23 3615 3630 4020 561138 513 528 TGGGTGTTCTGGAGTT Deoxy, MOE, and cEt 30 3617 3632 4021 561139 515 530 TCTGGGTGTTCTGGAG Deoxy, MOE, and cEt 24 3619 3634 4022 561140 517 532 CTTCTGGGTGTTCTGG Deoxy, MOE, and cEt 17 3621 3636 4023 561141 519 534 TACTTCTGGGTGTTCT Deoxy, MOE, and cEt 10 3623 3638 4024 561142 521 536 GTTACTTCTGGGTGTT Deoxy, MOE, and cEt 11 3625 3640 4025 561143 523 538 AAGTTACTTCTGGGTG Deoxy, MOE, and cEt 15 3627 3642 4026 560990 709 724 TTCTTGGTGCTCTTGG Deoxy, MOE, and cEt 79 6722 6737 111 561221 758 773 CCATCATGTTTTACAT Deoxy, MOE, and cEt 17 6771 6786 4027 561222 760 775 TGCCATCATGTTTTAC Deoxy, MOE, and cEt 22 N/A N/A 4028 561223 763 778 GAATGCCATCATGTTT Deoxy, MOE, and cEt 12 N/A N/A 4029 561224 765 780 AGGAATGCCATCATGT Deoxy, MOE, and cEt 26 N/A N/A 4030 561225 767 782 GCAGGAATGCCATCAT Deoxy, MOE, and cEt 32 N/A N/A 4031 561226 769 784 CAGCAGGAATGCCATC Deoxy, MOE, and cEt 29 N/A N/A 4032 561227 771 786 TTCAGCAGGAATGCCA Deoxy, MOE, and cEt 22 N/A N/A 4033 561228 773 788 CATTCAGCAGGAATGC Deoxy, MOE, and cEt 23 7358 7373 4034 561229 775 790 TACATTCAGCAGGAAT Deoxy, MOE, and cEt 28 7360 7375 4035 561230 777 792 GGTACATTCAGCAGGA Deoxy, MOE, and cEt 61 7362 7377 4036 561231 779 794 GTGGTACATTCAGCAG Deoxy, MOE, and cEt 57 7364 7379 4037 561232 781 796 TGGTGGTACATTCAGC Deoxy, MOE, and cEt 59 7366 7381 4038 561233 787 802 TATAAATGGTGGTACA Deoxy, MOE, and cEt 51 7372 7387 4039 561234 789 804 GTTATAAATGGTGGTA Deoxy, MOE, and cEt 50 7374 7389 4040 561235 791 806 CTGTTATAAATGGTGG Deoxy, MOE, and cEt 49 7376 7391 4041 561236 793 808 CTCTGTTATAAATGGT Deoxy, MOE, and cEt 39 7378 7393 4042 561237 795 810 ACCTCTGTTATAAATG Deoxy, MOE, and cEt 47 7380 7395 4043 561238 797 812 TCACCTCTGTTATAAA Deoxy, MOE, and cEt 44 7382 7397 4044 561239 799 814 GTTCACCTCTGTTATA Deoxy, MOE, and cEt 43 7384 7399 4045 561240 801 816 ATGTTCACCTCTGTTA Deoxy, MOE, and cEt 59 7386 7401 4046 561241 803 818 GTATGTTCACCTCTGT Deoxy, MOE, and cEt 69 7388 7403 164 337487 804 823 CACTTGTATGTTCACCTCTG 5-10-5 MOE 74 7389 7408 28 561242 805 820 TTGTATGTTCACCTCT Deoxy, MOE, and cEt 63 7390 7405 4047 561243 807 822 ACTTGTATGTTCACCT Deoxy, MOE, and cEt 63 7392 7407 4048 561244 809 824 CCACTTGTATGTTCAC Deoxy, MOE, and cEt 57 7394 7409 4049 561245 811 826 TGCCACTTGTATGTTC Deoxy, MOE, and cEt 36 7396 7411 4050 561246 813 828 CATGCCACTTGTATGT Deoxy, MOE, and cEt 33 7398 7413 4051 561247 815 830 TACATGCCACTTGTAT Deoxy, MOE, and cEt 37 7400 7415 4052 561248 817 832 CATACATGCCACTTGT Deoxy, MOE, and cEt 36 7402 7417 4053 561249 819 834 GGCATACATGCCACTT Deoxy, MOE, and cEt 20 7404 7419 4054 561250 821 836 ATGGCATACATGCCAC Deoxy, MOE, and cEt 0 7406 7421 4055 561251 823 838 TGATGGCATACATGCC Deoxy, MOE, and cEt 22 7408 7423 4056 561252 825 840 TCTGATGGCATACATG Deoxy, MOE, and cEt 34 7410 7425 4057 561253 827 842 GGTCTGATGGCATACA Deoxy, MOE, and cEt 46 7412 7427 4058 561254 829 844 TGGGTCTGATGGCATA Deoxy, MOE, and cEt 51 7414 7429 4059 561255 834 849 GTTGCTGGGTCTGATG Deoxy, MOE, and cEt 45 7419 7434 4060 561256 836 851 GAGTTGCTGGGTCTGA Deoxy, MOE, and cEt 70 7421 7436 165 561257 838 853 GAGAGTTGCTGGGTCT Deoxy, MOE, and cEt 57 7423 7438 4061 561258 840 855 TTGAGAGTTGCTGGGT Deoxy, MOE, and cEt 47 7425 7440 4062 561259 842 857 ACTTGAGAGTTGCTGG Deoxy, MOE, and cEt 53 7427 7442 4063 561260 844 859 AAACTTGAGAGTTGCT Deoxy, MOE, and cEt 71 7429 7444 166 561261 846 861 AAAAACTTGAGAGTTG Deoxy, MOE, and cEt 23 7431 7446 4064 561262 848 863 TGAAAAACTTGAGAGT Deoxy, MOE, and cEt 11 7433 7448 4065 561263 850 865 CATGAAAAACTTGAGA Deoxy, MOE, and cEt 34 7435 7450 4066 561264 852 867 GACATGAAAAACTTGA Deoxy, MOE, and cEt 25 7437 7452 4067 561265 860 875 TCACAGTAGACATGAA Deoxy, MOE, and cEt 16 7445 7460 4068 561266 862 877 CATCACAGTAGACATG Deoxy, MOE, and cEt 37 7447 7462 4069 561267 864 879 AACATCACAGTAGACA Deoxy, MOE, and cEt 57 7449 7464 4070 561268 866 881 ATAACATCACAGTAGA Deoxy, MOE, and cEt 40 7451 7466 4071 561269 868 883 ATATAACATCACAGTA Deoxy, MOE, and cEt 26 7453 7468 4072 561270 870 885 TGATATAACATCACAG Deoxy, MOE, and cEt 35 7455 7470 4073 561271 872 887 CCTGATATAACATCAC Deoxy, MOE, and cEt 60 7457 7472 4074 561272 874 889 TACCTGATATAACATC Deoxy, MOE, and cEt 37 7459 7474 4075 561273 876 891 ACTACCTGATATAACA Deoxy, MOE, and cEt 24 N/A N/A 4076 561274 878 893 GGACTACCTGATATAA Deoxy, MOE, and cEt 7 N/A N/A 4077 561275 880 895 ATGGACTACCTGATAT Deoxy, MOE, and cEt 33 N/A N/A 4078 561276 882 897 CCATGGACTACCTGAT Deoxy, MOE, and cEt 52 N/A N/A 4079 561277 884 899 GTCCATGGACTACCTG Deoxy, MOE, and cEt 71 7871 7886 167 561278 886 901 ATGTCCATGGACTACC Deoxy, MOE, and cEt 67 7873 7888 4080 561279 888 903 TAATGTCCATGGACTA Deoxy, MOE, and cEt 44 7875 7890 4081 559390 890 905 ATTAATGTCCATGGAC Deoxy, MOE, and cEt 28 7877 7892 4082 561280 892 907 GAATTAATGTCCATGG Deoxy, MOE, and cEt 51 7879 7894 4083 561281 894 909 TTGAATTAATGTCCAT Deoxy, MOE, and cEt 30 7881 7896 4084 561282 896 911 TGTTGAATTAATGTCC Deoxy, MOE, and cEt 38 7883 7898 4085 561283 898 913 GATGTTGAATTAATGT Deoxy, MOE, and cEt 11 7885 7900 4086 561284 900 915 TCGATGTTGAATTAAT Deoxy, MOE, and cEt 20 7887 7902 4087 561285 902 917 ATTCGATGTTGAATTA Deoxy, MOE, and cEt 12 7889 7904 4088 561286 904 919 CTATTCGATGTTGAAT Deoxy, MOE, and cEt 17 7891 7906 4089 561287 906 921 ATCTATTCGATGTTGA Deoxy, MOE, and cEt 32 7893 7908 4090 561288 908 923 CCATCTATTCGATGTT Deoxy, MOE, and cEt 69 7895 7910 168 561289 910 925 ATCCATCTATTCGATG Deoxy, MOE, and cEt 32 7897 7912 4091 561290 912 927 TGATCCATCTATTCGA Deoxy, MOE, and cEt 41 7899 7914 4092 561291 914 929 TGTGATCCATCTATTC Deoxy, MOE, and cEt 50 7901 7916 4093 561292 916 931 TTTGTGATCCATCTAT Deoxy, MOE, and cEt 50 7903 7918 4094 561293 918 933 GTTTTGTGATCCATCT Deoxy, MOE, and cEt 41 7905 7920 4095 561294 920 935 AAGTTTTGTGATCCAT Deoxy, MOE, and cEt 56 7907 7922 4096 561295 922 937 TGAAGTTTTGTGATCC Deoxy, MOE, and cEt 57 7909 7924 4097 561296 924 939 ATTGAAGTTTTGTGAT Deoxy, MOE, and cEt 0 7911 7926 4098 561450 1386 1401 CAACATTTTGGTTGAT Deoxy, MOE, and cEt 45 10358 10373 4099 561451 1389 1404 GATCAACATTTTGGTT Deoxy, MOE, and cEt 33 10361 10376 4100 561452 1391 1406 TGGATCAACATTTTGG Deoxy, MOE, and cEt 81 10363 10378 123 561453 1393 1408 GATGGATCAACATTTT Deoxy, MOE, and cEt 59 10365 10380 4101 561455 1397 1412 GTTGGATGGATCAACA Deoxy, MOE, and cEt 53 10369 10384 4102 561456 1399 1414 CTGTTGGATGGATCAA Deoxy, MOE, and cEt 71 10371 10386 4103 561457 1401 1416 ATCTGTTGGATGGATC Deoxy, MOE, and cEt 71 10373 10388 4104 561458 1403 1418 GAATCTGTTGGATGGA Deoxy, MOE, and cEt 84 10375 10390 124 561459 1405 1420 CTGAATCTGTTGGATG Deoxy, MOE, and cEt 72 10377 10392 4105 561460 1407 1422 TTCTGAATCTGTTGGA Deoxy, MOE, and cEt 78 10379 10394 125 561461 1414 1429 CAAAGCTTTCTGAATC Deoxy, MOE, and cEt 45 10386 10401 4106 561462 1421 1436 GTTCATTCAAAGCTTT Deoxy, MOE, and cEt 87 10393 10408 126 561463 1423 1438 CAGTTCATTCAAAGCT Deoxy, MOE, and cEt 85 10395 10410 127 561464 1425 1440 CTCAGTTCATTCAAAG Deoxy, MOE, and cEt 47 10397 10412 4107 561465 1427 1442 GCCTCAGTTCATTCAA Deoxy, MOE, and cEt 60 10399 10414 4108 561466 1429 1444 TTGCCTCAGTTCATTC Deoxy, MOE, and cEt 68 10401 10416 4109 561467 1431 1446 ATTTGCCTCAGTTCAT Deoxy, MOE, and cEt 61 10403 10418 4110 561468 1433 1448 AAATTTGCCTCAGTTC Deoxy, MOE, and cEt 48 10405 10420 4111 561469 1436 1451 TTTAAATTTGCCTCAG Deoxy, MOE, and cEt 59 10408 10423 4112 561470 1438 1453 CTTTTAAATTTGCCTC Deoxy, MOE, and cEt 50 10410 10425 4113 561471 1440 1455 GCCTTTTAAATTTGCC Deoxy, MOE, and cEt 73 10412 10427 4114 561472 1452 1467 GTTTAAATTATTGCCT Deoxy, MOE, and cEt 48 10424 10439 4115 561473 1463 1478 ATGAGGTTAATGTTTA Deoxy, MOE, and cEt 33 10435 10450 4116 561474 1465 1480 GAATGAGGTTAATGTT Deoxy, MOE, and cEt 29 10437 10452 4117 561475 1467 1482 TGGAATGAGGTTAATG Deoxy, MOE, and cEt 66 10439 10454 4118 561476 1469 1484 CTTGGAATGAGGTTAA Deoxy, MOE, and cEt 72 10441 10456 4119 561477 1471 1486 AACTTGGAATGAGGTT Deoxy, MOE, and cEt 69 10443 10458 4120 561478 1473 1488 TTAACTTGGAATGAGG Deoxy, MOE, and cEt 74 10445 10460 128 561479 1475 1490 CATTAACTTGGAATGA Deoxy, MOE, and cEt 5 10447 10462 4121 561480 1477 1492 CACATTAACTTGGAAT Deoxy, MOE, and cEt 26 10449 10464 4122 561481 1479 1494 ACCACATTAACTTGGA Deoxy, MOE, and cEt 59 10451 10466 4123 561482 1481 1496 AGACCACATTAACTTG Deoxy, MOE, and cEt 76 10453 10468 129 561483 1483 1498 TTAGACCACATTAACT Deoxy, MOE, and cEt 47 10455 10470 4124 561484 1485 1500 TATTAGACCACATTAA Deoxy, MOE, and cEt 38 10457 10472 4125 561485 1487 1502 ATTATTAGACCACATT Deoxy, MOE, and cEt 59 10459 10474 4126 561486 1489 1504 AGATTATTAGACCACA Deoxy, MOE, and cEt 84 10461 10476 130 561487 1491 1506 CCAGATTATTAGACCA Deoxy, MOE, and cEt 93 10463 10478 131 561488 1493 1508 TACCAGATTATTAGAC Deoxy, MOE, and cEt 22 10465 10480 4127 561489 1495 1510 AATACCAGATTATTAG Deoxy, MOE, and cEt 48 10467 10482 4128 561490 1497 1512 TTAATACCAGATTATT Deoxy, MOE, and cEt 22 10469 10484 4129 561491 1499 1514 ATTTAATACCAGATTA Deoxy, MOE, and cEt 14 10471 10486 4130 561492 1501 1516 GGATTTAATACCAGAT Deoxy, MOE, and cEt 74 10473 10488 4131 561493 1503 1518 AAGGATTTAATACCAG Deoxy, MOE, and cEt 70 10475 10490 4132 561494 1505 1520 TTAAGGATTTAATACC Deoxy, MOE, and cEt 14 10477 10492 4133 561495 1508 1523 CTCTTAAGGATTTAAT Deoxy, MOE, and cEt 12 10480 10495 4134 561496 1510 1525 TTCTCTTAAGGATTTA Deoxy, MOE, and cEt 47 10482 10497 4135 561497 1513 1528 GCTTTCTCTTAAGGAT Deoxy, MOE, and cEt 73 10485 10500 4136 561498 1515 1530 AAGCTTTCTCTTAAGG Deoxy, MOE, and cEt 59 10487 10502 4137 561499 1517 1532 TCAAGCTTTCTCTTAA Deoxy, MOE, and cEt 62 10489 10504 4138 561500 1526 1541 ATCTATTTCTCAAGCT Deoxy, MOE, and cEt 76 10498 10513 132 561501 1547 1562 AGTGACTTTAAGATAA Deoxy, MOE, and cEt 23 10519 10534 4139 561502 1549 1564 ACAGTGACTTTAAGAT Deoxy, MOE, and cEt 62 10521 10536 4140 561503 1551 1566 AGACAGTGACTTTAAG Deoxy, MOE, and cEt 55 10523 10538 4141 561504 1553 1568 ATAGACAGTGACTTTA Deoxy, MOE, and cEt 74 10525 10540 133 561505 1555 1570 AAATAGACAGTGACTT Deoxy, MOE, and cEt 59 10527 10542 4142 561506 1557 1572 TTAAATAGACAGTGAC Deoxy, MOE, and cEt 38 10529 10544 4143 561507 1559 1574 TCTTAAATAGACAGTG Deoxy, MOE, and cEt 54 10531 10546 4144 561508 1561 1576 AATCTTAAATAGACAG Deoxy, MOE, and cEt 22 10533 10548 4145 561509 1563 1578 TTAATCTTAAATAGAC Deoxy, MOE, and cEt 0 10535 10550 4146 561510 1565 1580 GTTTAATCTTAAATAG Deoxy, MOE, and cEt 0 10537 10552 4147 561511 1569 1584 GTATGTTTAATCTTAA Deoxy, MOE, and cEt 13 10541 10556 4148 561512 1572 1587 ATTGTATGTTTAATCT Deoxy, MOE, and cEt 40 10544 10559 4149 561513 1575 1590 GTGATTGTATGTTTAA Deoxy, MOE, and cEt 71 10547 10562 4150 561514 1578 1593 TATGTGATTGTATGTT Deoxy, MOE, and cEt 58 10550 10565 4151 561515 1580 1595 GTTATGTGATTGTATG Deoxy, MOE, and cEt 68 10552 10567 4152 561516 1582 1597 AGGTTATGTGATTGTA Deoxy, MOE, and cEt 73 10554 10569 4153 561517 1584 1599 TAAGGTTATGTGATTG Deoxy, MOE, and cEt 64 10556 10571 4154 561518 1586 1601 TTTAAGGTTATGTGAT Deoxy, MOE, and cEt 0 10558 10573 4155 561519 1588 1603 TCTTTAAGGTTATGTG Deoxy, MOE, and cEt 53 10560 10575 4156 561520 1590 1605 ATTCTTTAAGGTTATG Deoxy, MOE, and cEt 29 10562 10577 4157 561521 1592 1607 GTATTCTTTAAGGTTA Deoxy, MOE, and cEt 24 10564 10579 4158 561522 1594 1609 CGGTATTCTTTAAGGT Deoxy, MOE, and cEt 70 10566 10581 4159 561523 1596 1611 AACGGTATTCTTTAAG Deoxy, MOE, and cEt 42 10568 10583 4160 561524 1598 1613 TAAACGGTATTCTTTA Deoxy, MOE, and cEt 26 10570 10585 4161 561525 1600 1615 TGTAAACGGTATTCTT Deoxy, MOE, and cEt 59 10572 10587 4162 561526 1602 1617 AATGTAAACGGTATTC Deoxy, MOE, and cEt 57 10574 10589 4142

TABLE 153 Inhibition of ANGPTL3 mRNA by oligonucleotides targeting SEQ ID NO: 1 and 2 SEQ ID SEQ ID NO: 1 NO: SEQ ID SEQ ID Start 1 Stop % NO: 2 NO: 2 SEQ ISIS NO Site Site Sequence Chemistry inhibition Start Site Stop Site ID NO 561681 N/A N/A TCTGGAAGCAGACCTA Deoxy, MOE, and cEt 37 3096 3111 4164 561682 N/A N/A CTTCTGGAAGCAGACC Deoxy, MOE, and cEt 27 3098 3113 4165 561683 N/A N/A AAATAAGGTATAGTGA Deoxy, MOE, and cEt 2 11084 11099 4166 561684 N/A N/A TAGTATTAAGTGTTAA Deoxy, MOE, and cEt 14 11133 11148 4167 561685 N/A N/A TCATAGTATTAAGTGT Deoxy, MOE, and cEt 0 11136 11151 4168 561686 N/A N/A AGATTCCTTTACAATT Deoxy, MOE, and cEt 21 11160 11175 4169 561687 N/A N/A ACAAGATTCCTTTACA Deoxy, MOE, and cEt 21 11163 11178 4170 561688 N/A N/A CTGACAAGATTCCTTT Deoxy, MOE, and cEt 70 11166 11181 4171 561689 N/A N/A AATCTGACAAGATTCC Deoxy, MOE, and cEt 83 11169 11184 180 561690 N/A N/A TGTAATCTGACAAGAT Deoxy, MOE, and cEt 46 11172 11187 4172 561691 N/A N/A TACTGTAATCTGACAA Deoxy, MOE, and cEt 47 11175 11190 4173 561692 N/A N/A TCTTACTGTAATCTGA Deoxy, MOE, and cEt 50 11178 11193 4174 561693 N/A N/A CATTCTTACTGTAATC Deoxy, MOE, and cEt 40 11181 11196 4175 561694 N/A N/A GTTCATTCTTACTGTA Deoxy, MOE, and cEt 71 11184 11199 4176 561695 N/A N/A ATATGTTCATTCTTAC Deoxy, MOE, and cEt 2 11188 11203 4177 561696 N/A N/A GCCACAAATATGTTCA Deoxy, MOE, and cEt 80 11195 11210 4178 561697 N/A N/A GATGCCACAAATATGT Deoxy, MOE, and cEt 70 11198 11213 4179 561698 N/A N/A CTCGATGCCACAAATA Deoxy, MOE, and cEt 80 11201 11216 181 561699 N/A N/A TAACTCGATGCCACAA Deoxy, MOE, and cEt 86 11204 11219 182 561700 N/A N/A CTTTAACTCGATGCCA Deoxy, MOE, and cEt 77 11207 11222 4180 561701 N/A N/A AAACTTTAACTCGATG Deoxy, MOE, and cEt 39 11210 11225 4181 561702 N/A N/A TATAAACTTTAACTCG Deoxy, MOE, and cEt 13 11213 11228 4182 561703 N/A N/A CACAGCATATTTAGGG Deoxy, MOE, and cEt 71 11233 11248 4183 561704 N/A N/A TAGAATCACAGCATAT Deoxy, MOE, and cEt 68 11239 11254 4184 561705 N/A N/A TATTAGAATCACAGCA Deoxy, MOE, and cEt 73 11242 11257 4185 561706 N/A N/A AATGTATTAGAATCAC Deoxy, MOE, and cEt 40 11246 11261 4186 561707 N/A N/A ACGAATGTATTAGAAT Deoxy, MOE, and cEt 22 11249 11264 4187 561708 N/A N/A TACACGAATGTATTAG Deoxy, MOE, and cEt 33 11252 11267 4188 561709 N/A N/A ACCTACACGAATGTAT Deoxy, MOE, and cEt 42 11255 11270 4189 561710 N/A N/A AAAACCTACACGAATG Deoxy, MOE, and cEt 24 11258 11273 4190 561711 N/A N/A TTGAAAACCTACACGA Deoxy, MOE, and cEt 34 11261 11276 4191 561712 N/A N/A TACTTGAAAACCTACA Deoxy, MOE, and cEt 33 11264 11279 4192 561713 N/A N/A GTTTATTTCTACTTGA Deoxy, MOE, and cEt 53 11273 11288 4193 561714 N/A N/A GAGGTTTATTTCTACT Deoxy, MOE, and cEt 69 11276 11291 4194 561715 N/A N/A TACGAGGTTTATTTCT Deoxy, MOE, and cEt 21 11279 11294 4195 561716 N/A N/A TGTTACGAGGTTTATT Deoxy, MOE, and cEt 47 11282 11297 4196 561717 N/A N/A ACTTGTTACGAGGTTT Deoxy, MOE, and cEt 70 11285 11300 4197 561718 N/A N/A CAGTAACTTGTTACGA Deoxy, MOE, and cEt 60 11290 11305 4198 561719 N/A N/A GTTCAGTAACTTGTTA Deoxy, MOE, and cEt 40 11293 11308 4199 561720 N/A N/A TCAGGCTGTTTAAACG Deoxy, MOE, and cEt 59 11308 11323 4200 561721 N/A N/A TTGTCAGGCTGTTTAA Deoxy, MOE, and cEt 74 11311 11326 4201 561722 N/A N/A TGCTTGTCAGGCTGTT Deoxy, MOE, and cEt 82 11314 11329 183 561723 N/A N/A ACATGCTTGTCAGGCT Deoxy, MOE, and cEt 84 11317 11332 184 561724 N/A N/A TATACATGCTTGTCAG Deoxy, MOE, and cEt 75 11320 11335 4202 561725 N/A N/A GTCTTTGTTTATTGAA Deoxy, MOE, and cEt 49 11347 11362 4203 561726 N/A N/A TGGGTCTTTGTTTATT Deoxy, MOE, and cEt 27 11350 11365 4204 561727 N/A N/A GACTGGGTCTTTGTTT Deoxy, MOE, and cEt 20 11353 11368 4205 561728 N/A N/A ATAATTTAGGGACTGG Deoxy, MOE, and cEt 20 11363 11378 4206 561729 N/A N/A TCTATAATTTAGGGAC Deoxy, MOE, and cEt 39 11366 11381 4207 561730 N/A N/A CGATAAACATGCAAGA Deoxy, MOE, and cEt 68 11394 11409 4208 561731 N/A N/A TGTCGATAAACATGCA Deoxy, MOE, and cEt 80 11397 11412 4209 561732 N/A N/A TGATGTCGATAAACAT Deoxy, MOE, and cEt 68 11400 11415 4210 561733 N/A N/A TTGTGATGTCGATAAA Deoxy, MOE, and cEt 28 11403 11418 4211 561734 N/A N/A CTGTTGTGATGTCGAT Deoxy, MOE, and cEt 74 11406 11421 4212 561735 N/A N/A GATCTGTTGTGATGTC Deoxy, MOE, and cEt 59 11409 11424 4213 561736 N/A N/A AGGGATCTGTTGTGAT Deoxy, MOE, and cEt 24 11412 11427 4214 561737 N/A N/A TTTAGGGATCTGTTGT Deoxy, MOE, and cEt 19 11415 11430 4215 561738 N/A N/A GGATTTAGGGATCTGT Deoxy, MOE, and cEt 27 11418 11433 4216 561739 N/A N/A GATTTAGGGATTTAGG Deoxy, MOE, and cEt 44 11425 11440 4217 561740 N/A N/A TCTTTAGGGATTTAGG Deoxy, MOE, and cEt 38 11433 11448 4218 561741 N/A N/A TAATCTTTAGGGATTT Deoxy, MOE, and cEt 0 11436 11451 4219 561742 N/A N/A ATCTAATCTTTAGGGA Deoxy, MOE, and cEt 0 11439 11454 4220 561743 N/A N/A TGTATCTAATCTTTAG Deoxy, MOE, and cEt 15 11442 11457 4221 561744 N/A N/A AAATTTGTATCTAATC Deoxy, MOE, and cEt 21 11447 11462 4222 561745 N/A N/A GTAAAAAATTTGTATC Deoxy, MOE, and cEt 23 11452 11467 4223 561746 N/A N/A GTGGTAAAAAATTTGT Deoxy, MOE, and cEt 32 11455 11470 4224 561747 N/A N/A GATACTGTGGTAAAAA Deoxy, MOE, and cEt 45 11461 11476 4225 561748 N/A N/A AGTGATACTGTGGTAA Deoxy, MOE, and cEt 60 11464 11479 4226 561749 N/A N/A ACAAGTGATACTGTGG Deoxy, MOE, and cEt 75 11467 11482 4227 561750 N/A N/A CTGACAAGTGATACTG Deoxy, MOE, and cEt 59 11470 11485 4228 561751 N/A N/A ATTCTGACAAGTGATA Deoxy, MOE, and cEt 48 11473 11488 4229 561752 N/A N/A TAAATTCTGACAAGTG Deoxy, MOE, and cEt 59 11476 11491 4230 561753 N/A N/A TACTGGCAGTTTTAAA Deoxy, MOE, and cEt 42 11508 11523 4231 561754 N/A N/A TCTTACTGGCAGTTTT Deoxy, MOE, and cEt 51 11511 11526 4232 561755 N/A N/A ATTTCTTACTGGCAGT Deoxy, MOE, and cEt 69 11514 11529 4233 561756 N/A N/A AAAATTTCTTACTGGC Deoxy, MOE, and cEt 57 11517 11532 4234 561757 N/A N/A AACAAATGGGTTTAAT Deoxy, MOE, and cEt 0 11535 11550 4235 562374 N/A N/A GAATATTTGCAAGTCT Deoxy, MOE, and cEt 68 9230 9245 4236 562375 N/A N/A GTAGAGGAATATTTGC Deoxy, MOE, and cEt 83 9236 9251 151 562376 N/A N/A TCATTGGTAGAGGAAT Deoxy, MOE, and cEt 23 9242 9257 4237 562377 N/A N/A ATATTTTAAAGTCTCG Deoxy, MOE, and cEt 17 9258 9273 4238 562378 N/A N/A GTTACATTATTATAGA Deoxy, MOE, and cEt 29 9273 9288 4239 562379 N/A N/A GTGAAATGTGTTACAT Deoxy, MOE, and cEt 54 9282 9297 4240 562380 N/A N/A TCACCAGTGAAATGTG Deoxy, MOE, and cEt 64 9288 9303 4241 562381 N/A N/A CATGTTTCACCAGTGA Deoxy, MOE, and cEt 78 9294 9309 4242 562382 N/A N/A ACAAGACATGTTTCAC Deoxy, MOE, and cEt 36 9300 9315 4243 562383 N/A N/A CATATGACAAGACATG Deoxy, MOE, and cEt 42 9306 9321 4244 562384 N/A N/A CTATAATGCATATGAC Deoxy, MOE, and cEt 5 9314 9329 4245 562385 N/A N/A TCCTTTCTATAATGCA Deoxy, MOE, and cEt 65 9320 9335 4246 562386 N/A N/A TGATTATCCTTTCTAT Deoxy, MOE, and cEt 27 9326 9341 4247 562387 N/A N/A AAAGTCTGATTATCCT Deoxy, MOE, and cEt 90 9332 9347 152 562388 N/A N/A TAACTGAAAGTCTGAT Deoxy, MOE, and cEt 59 9338 9353 4248 562389 N/A N/A GTGCACAAAAATGTTA Deoxy, MOE, and cEt 42 9366 9381 4249 562390 N/A N/A AGCTATGTGCACAAAA Deoxy, MOE, and cEt 77 9372 9387 4250 562391 N/A N/A GAAGATAGCTATGTGC Deoxy, MOE, and cEt 64 9378 9393 4251 562392 N/A N/A TTTATTGAAGATAGCT Deoxy, MOE, and cEt 33 9384 9399 4252 562393 N/A N/A TCATTTTAGTGTATCT Deoxy, MOE, and cEt 40 9424 9439 4253 562394 N/A N/A CCTTGATCATTTTAGT Deoxy, MOE, and cEt 15 9430 9445 4254 562395 N/A N/A TGAATCCCTTGATCAT Deoxy, MOE, and cEt 59 9436 9451 4255 562396 N/A N/A TAGTCTTGAATCCCTT Deoxy, MOE, and cEt 83 9442 9457 153 562397 N/A N/A GTTGTTTAGTCTTGAA Deoxy, MOE, and cEt 65 9448 9463 4256 562398 N/A N/A AATTGAGTTGTTTAGT Deoxy, MOE, and cEt 21 9454 9469 4257 562399 N/A N/A GCAACTAATTGAGTTG Deoxy, MOE, and cEt 15 9460 9475 4258 562400 N/A N/A ATTGGTGCAACTAATT Deoxy, MOE, and cEt 25 9466 9481 4259 562401 N/A N/A GTTTTTTATTGGTGCA Deoxy, MOE, and cEt 53 9473 9488 4260 562402 N/A N/A GGACACTGACAGTTTT Deoxy, MOE, and cEt 43 9496 9511 4261 562403 N/A N/A CAGGTTGGACACTGAC Deoxy, MOE, and cEt 23 9502 9517 4262 562404 N/A N/A TAAGTACAGGTTGGAC Deoxy, MOE, and cEt 33 9508 9523 4263 562405 N/A N/A AGTTATTAAGTACAGG Deoxy, MOE, and cEt 34 9514 9529 4264 562406 N/A N/A TCTGTGAGTTATTAAG Deoxy, MOE, and cEt 10 9520 9535 4265 562407 N/A N/A ACCAAAATTCTCCTGA Deoxy, MOE, and cEt 1 9554 9569 4266 562408 N/A N/A ACCTGAATAACCCTCT Deoxy, MOE, and cEt 73 9811 9826 4267 562409 N/A N/A GGTATCAGAAAAAGAT Deoxy, MOE, and cEt 14 9827 9842 4268 562410 N/A N/A AGTATTGGTATCAGAA Deoxy, MOE, and cEt 13 9833 9848 4269 562411 N/A N/A GGAAGATACTTTGAAG Deoxy, MOE, and cEt 25 9861 9876 4270 562412 N/A N/A AATGTGGGAAGATACT Deoxy, MOE, and cEt 23 9867 9882 4271 562413 N/A N/A CAGATAATAGCTAATA Deoxy, MOE, and cEt 29 9882 9897 4272 562414 N/A N/A TCATTGCAGATAATAG Deoxy, MOE, and cEt 45 9888 9903 4273 562415 N/A N/A AAGTTGTCATTGCAGA Deoxy, MOE, and cEt 86 9894 9909 154 562416 N/A N/A GATTCGGATTTTTAAA Deoxy, MOE, and cEt 19 9909 9924 4274 562417 N/A N/A ATTTGGGATTCGGATT Deoxy, MOE, and cEt 34 9915 9930 4275 562418 N/A N/A ACGCTTATTTGGGATT Deoxy, MOE, and cEt 64 9921 9936 4276 562419 N/A N/A TCTAGAGAGAAAACGC Deoxy, MOE, and cEt 64 9933 9948 4277 562420 N/A N/A AGTTAAGAGGTTTTCG Deoxy, MOE, and cEt 34 9949 9964 4278 562421 N/A N/A CATTATAGTTAAGAGG Deoxy, MOE, and cEt 24 9955 9970 4279 562422 N/A N/A CACTTTCATTATAGTT Deoxy, MOE, and cEt 13 9961 9976 4280 562423 N/A N/A TAGAATGAACACTTTC Deoxy, MOE, and cEt 63 9970 9985 4281 562424 N/A N/A TTGAACTAGAATGAAC Deoxy, MOE, and cEt 16 9976 9991 4282 562425 N/A N/A ACCTGATTGAACTAGA Deoxy, MOE, and cEt 51 9982 9997 4283 562426 N/A N/A TAAAATACCTGATTGA Deoxy, MOE, and cEt 19 9988 10003 4284 562427 N/A N/A TAGAGGTAAAATACCT Deoxy, MOE, and cEt 12 9994 10009 4285 562428 N/A N/A GAAGATTAGAGGTAAA Deoxy, MOE, and cEt 1 10000 10015 4286 562429 N/A N/A TCTGAGGAAGATTAGA Deoxy, MOE, and cEt 31 10006 10021 4287 562430 N/A N/A TATACACTACCAAAAA Deoxy, MOE, and cEt 0 10030 10045 4288 562431 N/A N/A ATAATCTATACACTAC Deoxy, MOE, and cEt 0 10036 10051 4289 562432 N/A N/A TAAGTCCCAATTTTAA Deoxy, MOE, and cEt 33 10065 10080 4290 562433 N/A N/A TCTGTATAAGTCCCAA Deoxy, MOE, and cEt 89 10071 10086 155 562434 N/A N/A CCAGTTTTAAATAATC Deoxy, MOE, and cEt 20 10085 10100 4291 562435 N/A N/A TGTATCCCAGTTTTAA Deoxy, MOE, and cEt 44 10091 10106 4292 562436 N/A N/A GATGCATGTATCCCAG Deoxy, MOE, and cEt 91 10097 10112 156 562437 N/A N/A GTTTTAGATGCATGTA Deoxy, MOE, and cEt 69 10103 10118 4293 562438 N/A N/A TACAGTGTTTTAGATG Deoxy, MOE, and cEt 28 10109 10124 4294 562439 N/A N/A GTAAGTTTATCTTCCT Deoxy, MOE, and cEt 78 10138 10153 157 562440 N/A N/A TTCCCCGTAAGTTTAT Deoxy, MOE, and cEt 33 10144 10159 4295 562441 N/A N/A CTGTATTTCCCCGTAA Deoxy, MOE, and cEt 55 10150 10165 4296 562442 N/A N/A CTGTTACTGTATTTCC Deoxy, MOE, and cEt 79 10156 10171 158 562443 N/A N/A TAGTTACTGTTACTGT Deoxy, MOE, and cEt 70 10162 10177 4297 562444 N/A N/A CGTATGTAGTTACTGT Deoxy, MOE, and cEt 66 10168 10183 4298 562445 N/A N/A AATGGGTACAGACTCG Deoxy, MOE, and cEt 72 10182 10197 4299 562446 N/A N/A GCAATTTAATGGGTAC Deoxy, MOE, and cEt 59 10189 10204 4300 562447 N/A N/A GATAGATATGCAATTT Deoxy, MOE, and cEt 20 10198 10213 4301 562448 N/A N/A AAAGGAGATAGATATG Deoxy, MOE, and cEt 22 10204 10219 4302 562449 N/A N/A CCTCCTAAAGGAGATA Deoxy, MOE, and cEt 42 10210 10225 4303 562450 N/A N/A CACCAGCCTCCTAAAG Deoxy, MOE, and cEt 37 10216 10231 4304 544120 707 726 AGTTCTTGGTGCTCTTGGCT 5-10-5 MOE 83 6720 6739 15 560990 709 724 TTCTTGGTGCTCTTGG Deoxy, MOE, and cEt 89 6722 6737 111 337487 804 823 CACTTGTATGTTCACCTCTG 5-10-5 MOE 81 7389 7408 28 561373 1197 1212 TTTGTGATCCCAAGTA Deoxy, MOE, and cEt 40 9772 9787 4305 561374 1199 1214 GCTTTGTGATCCCAAG Deoxy, MOE, and cEt 76 9774 9789 4306 561375 1201 1216 TTGCTTTGTGATCCCA Deoxy, MOE, and cEt 82 9776 9791 4307 561376 1203 1218 TTTTGCTTTGTGATCC Deoxy, MOE, and cEt 40 9778 9793 4308 561377 1205 1220 CCTTTTGCTTTGTGAT Deoxy, MOE, and cEt 38 9780 9795 4309 561378 1207 1222 GTCCTTTTGCTTTGTG Deoxy, MOE, and cEt 75 9782 9797 4310 561379 1209 1224 GTGTCCTTTTGCTTTG Deoxy, MOE, and cEt 40 9784 9799 4311 561380 1212 1227 GAAGTGTCCTTTTGCT Deoxy, MOE, and cEt 23 9787 9802 4312 561381 1214 1229 TTGAAGTGTCCTTTTG Deoxy, MOE, and cEt 26 9789 9804 4313 561382 1216 1231 AGTTGAAGTGTCCTTT Deoxy, MOE, and cEt 34 9791 9806 4314 561383 1218 1233 ACAGTTGAAGTGTCCT Deoxy, MOE, and cEt 27 9793 9808 4315 561384 1220 1235 GGACAGTTGAAGTGTC Deoxy, MOE, and cEt 19 9795 9810 4316 561385 1222 1237 CTGGACAGTTGAAGTG Deoxy, MOE, and cEt 34 9797 9812 4317 561386 1224 1239 CTCTGGACAGTTGAAG Deoxy, MOE, and cEt 19 9799 9814 4318 561387 1226 1241 CCCTCTGGACAGTTGA Deoxy, MOE, and cEt 54 9801 9816 4319 561388 1228 1243 AACCCTCTGGACAGTT Deoxy, MOE, and cEt 50 9803 9818 4320 561389 1230 1245 ATAACCCTCTGGACAG Deoxy, MOE, and cEt 35 9805 9820 4321 561390 1232 1247 GAATAACCCTCTGGAC Deoxy, MOE, and cEt 34 9807 9822 4322 561391 1234 1249 CTGAATAACCCTCTGG Deoxy, MOE, and cEt 62 9809 9824 4323 561392 1236 1251 TCCTGAATAACCCTCT Deoxy, MOE, and cEt 57 N/A N/A 4324 561393 1238 1253 CCTCCTGAATAACCCT Deoxy, MOE, and cEt 30 N/A N/A 4325 561394 1246 1261 ACCACCAGCCTCCTGA Deoxy, MOE, and cEt 70 N/A N/A 4326 561395 1251 1266 ATGCCACCACCAGCCT Deoxy, MOE, and cEt 68 10223 10238 4327 561396 1253 1268 TCATGCCACCACCAGC Deoxy, MOE, and cEt 72 10225 10240 4328 561397 1255 1270 CATCATGCCACCACCA Deoxy, MOE, and cEt 67 10227 10242 4329 561398 1257 1272 CTCATCATGCCACCAC Deoxy, MOE, and cEt 77 10229 10244 172 561399 1259 1274 CACTCATCATGCCACC Deoxy, MOE, and cEt 74 10231 10246 2330 561400 1261 1276 CACACTCATCATGCCA Deoxy, MOE, and cEt 80 10233 10248 173 561401 1263 1278 TCCACACTCATCATGC Deoxy, MOE, and cEt 64 10235 10250 4331 561402 1265 1280 TCTCCACACTCATCAT Deoxy, MOE, and cEt 42 10237 10252 4332 561403 1267 1282 TTTCTCCACACTCATC Deoxy, MOE, and cEt 47 10239 10254 4333 561404 1269 1284 GTTTTCTCCACACTCA Deoxy, MOE, and cEt 77 10241 10256 4334 561405 1272 1287 GTTGTTTTCTCCACAC Deoxy, MOE, and cEt 53 10244 10259 4335 561406 1274 1289 AGGTTGTTTTCTCCAC Deoxy, MOE, and cEt 67 10246 10261 4336 561407 1276 1291 TTAGGTTGTTTTCTCC Deoxy, MOE, and cEt 73 10248 10263 4337 561408 1282 1297 TACCATTTAGGTTGTT Deoxy, MOE, and cEt 30 10254 10269 4338 561409 1284 1299 TTTACCATTTAGGTTG Deoxy, MOE, and cEt 22 10256 10271 4339 561410 1286 1301 TATTTACCATTTAGGT Deoxy, MOE, and cEt 24 10258 10273 4340 561411 1292 1307 TTGTTATATTTACCAT Deoxy, MOE, and cEt 41 10264 10279 4341 561412 1294 1309 GTTTGTTATATTTACC Deoxy, MOE, and cEt 37 10266 10281 4342 561413 1298 1313 CTTGGTTTGTTATATT Deoxy, MOE, and cEt 45 10270 10285 4343 561414 1300 1315 CTCTTGGTTTGTTATA Deoxy, MOE, and cEt 73 10272 10287 4344 561415 1302 1317 TGCTCTTGGTTTGTTA Deoxy, MOE, and cEt 45 10274 10289 4345 561416 1304 1319 TTTGCTCTTGGTTTGT Deoxy, MOE, and cEt 67 10276 10291 4346 561417 1307 1322 GATTTTGCTCTTGGTT Deoxy, MOE, and cEt 75 10279 10294 4347 561418 1309 1324 TAGATTTTGCTCTTGG Deoxy, MOE, and cEt 87 10281 10296 169 561419 1311 1326 CTTAGATTTTGCTCTT Deoxy, MOE, and cEt 64 10283 10298 4348 561420 1313 1328 GGCTTAGATTTTGCTC Deoxy, MOE, and cEt 58 10285 10300 4349 561421 1315 1330 CTGGCTTAGATTTTGC Deoxy, MOE, and cEt 70 10287 10302 4350 561422 1317 1332 CTCTGGCTTAGATTTT Deoxy, MOE, and cEt 38 10289 10304 4351 561423 1319 1334 CTCTCTGGCTTAGATT Deoxy, MOE, and cEt 63 10291 10306 4352 561424 1321 1336 TCCTCTCTGGCTTAGA Deoxy, MOE, and cEt 76 10293 10308 4353 561425 1323 1338 TCTCCTCTCTGGCTTA Deoxy, MOE, and cEt 67 10295 10310 4354 561426 1332 1347 TAATCCTCTTCTCCTC Deoxy, MOE, and cEt 50 10304 10319 4355 561427 1334 1349 GATAATCCTCTTCTCC Deoxy, MOE, and cEt 36 10306 10321 4356 561428 1336 1351 AAGATAATCCTCTTCT Deoxy, MOE, and cEt 43 10308 10323 4357 561429 1338 1353 CCAAGATAATCCTCTT Deoxy, MOE, and cEt 59 10310 10325 4358 561430 1340 1355 TTCCAAGATAATCCTC Deoxy, MOE, and cEt 65 10312 10327 4359 561431 1342 1357 ACTTCCAAGATAATCC Deoxy, MOE, and cEt 74 10314 10329 4360 561432 1344 1359 AGACTTCCAAGATAAT Deoxy, MOE, and cEt 52 10316 10331 4361 561433 1346 1361 TGAGACTTCCAAGATA Deoxy, MOE, and cEt 49 10318 10333 4362 561434 1348 1363 TTTGAGACTTCCAAGA Deoxy, MOE, and cEt 47 10320 10335 4363 561435 1350 1365 ATTTTGAGACTTCCAA Deoxy, MOE, and cEt 64 10322 10337 4364 561436 1352 1367 CCATTTTGAGACTTCC Deoxy, MOE, and cEt 84 10324 10339 170 561437 1354 1369 TTCCATTTTGAGACTT Deoxy, MOE, and cEt 67 10326 10341 4365 561438 1356 1371 CCTTCCATTTTGAGAC Deoxy, MOE, and cEt 53 10328 10343 4366 561439 1358 1373 AACCTTCCATTTTGAG Deoxy, MOE, and cEt 37 10330 10345 4367 561440 1360 1375 ATAACCTTCCATTTTG Deoxy, MOE, and cEt 50 10332 10347 4368 561441 1362 1377 GTATAACCTTCCATTT Deoxy, MOE, and cEt 27 10334 10349 4369 561442 1364 1379 GAGTATAACCTTCCAT Deoxy, MOE, and cEt 65 10336 10351 4370 561443 1366 1381 TAGAGTATAACCTTCC Deoxy, MOE, and cEt 84 10338 10353 171 561444 1368 1383 TATAGAGTATAACCTT Deoxy, MOE, and cEt 17 10340 10355 4371 561445 1370 1385 TTTATAGAGTATAACC Deoxy, MOE, and cEt 37 10342 10357 4372 561446 1373 1388 GATTTTATAGAGTATA Deoxy, MOE, and cEt 28 10345 10360 4373 561447 1375 1390 TTGATTTTATAGAGTA Deoxy, MOE, and cEt 21 10347 10362 4374 561448 1377 1392 GGTTGATTTTATAGAG Deoxy, MOE, and cEt 28 10349 10364 4375 561449 1379 1394 TTGGTTGATTTTATAG Deoxy, MOE, and cEt 22 10351 10366 4376 567295 1452 1471 TAATGTTTAAATTATTGCCT 5-10-5 MOE 43 10424 10443 4377 567296 1455 1474 GGTTAATGTTTAAATTATTG 5-10-5 MOE 22 10427 10446 4378 567297 1456 1475 AGGTTAATGTTTAAATTATT 5-10-5 MOE 0 10428 10447 4379 567298 1457 1476 GAGGTTAATGTTTAAATTAT 5-10-5 MOE 0 10429 10448 4380 567299 1458 1477 TGAGGTTAATGTTTAAATTA 5-10-5 MOE 6 10430 10449 4381 567300 1460 1479 AATGAGGTTAATGTTTAAAT 5-10-5 MOE 14 10432 10451 4382 567301 1461 1480 GAATGAGGTTAATGTTTAAA 5-10-5 MOE 5 10433 10452 4383 567302 1462 1481 GGAATGAGGTTAATGTTTAA 5-10-5 MOE 27 10434 10453 4384 567303 1463 1482 TGGAATGAGGTTAATGTTTA 5-10-5 MOE 32 10435 10454 4385 567304 1464 1483 TTGGAATGAGGTTAATGTTT 5-10-5 MOE 37 10436 10455 4386 567305 1465 1484 CTTGGAATGAGGTTAATGTT 5-10-5 MOE 25 10437 10456 4387 567306 1468 1487 TAACTTGGAATGAGGTTAAT 5-10-5 MOE 29 10440 10459 4388 567307 1469 1488 TTAACTTGGAATGAGGTTAA 5-10-5 MOE 44 10441 10460 4389 337513 1470 1489 ATTAACTTGGAATGAGGTTA 5-10-5 MOE 52 10442 10461 4390 567308 1471 1490 CATTAACTTGGAATGAGGTT 5-10-5 MOE 62 10443 10462 4391 567309 1472 1491 ACATTAACTTGGAATGAGGT 5-10-5 MOE 58 10444 10463 4392 567310 1473 1492 CACATTAACTTGGAATGAGG 5-10-5 MOE 78 10445 10464 92 567311 1475 1494 ACCACATTAACTTGGAATGA 5-10-5 MOE 59 10447 10466 4393 567312 1476 1495 GACCACATTAACTTGGAATG 5-10-5 MOE 57 10448 10467 4394 337514 1477 1496 AGACCACATTAACTTGGAAT 5-10-5 MOE 71 10449 10468 4395 567313 1478 1497 TAGACCACATTAACTTGGAA 5-10-5 MOE 43 10450 10469 4396 567314 1479 1498 TTAGACCACATTAACTTGGA 5-10-5 MOE 59 10451 10470 4397 567315 1480 1499 ATTAGACCACATTAACTTGG 5-10-5 MOE 70 10452 10471 4398 567316 1481 1500 TATTAGACCACATTAACTTG 5-10-5 MOE 53 10453 10472 4399 567317 1482 1501 TTATTAGACCACATTAACTT 5-10-5 MOE 49 10454 10473 4400 567318 1483 1502 ATTATTAGACCACATTAACT 5-10-5 MOE 41 10455 10474 4401 567319 1484 1503 GATTATTAGACCACATTAAC 5-10-5 MOE 47 10456 10475 4402 567320 1487 1506 CCAGATTATTAGACCACATT 5-10-5 MOE 86 10459 10478 93 567321 1489 1508 TACCAGATTATTAGACCACA 5-10-5 MOE 85 10461 10480 94 337516 1490 1509 ATACCAGATTATTAGACCAC 5-10-5 MOE 77 10462 10481 86 567322 1491 1510 AATACCAGATTATTAGACCA 5-10-5 MOE 50 10463 10482 4403 567323 1492 1511 TAATACCAGATTATTAGACC 5-10-5 MOE 56 10464 10483 4404 567324 1494 1513 TTTAATACCAGATTATTAGA 5-10-5 MOE 9 10466 10485 4405 567325 1495 1514 ATTTAATACCAGATTATTAG 5-10-5 MOE 24 10467 10486 4406 567326 1496 1515 GATTTAATACCAGATTATTA 5-10-5 MOE 37 10468 10487 4407 567327 1500 1519 TAAGGATTTAATACCAGATT 5-10-5 MOE 60 10472 10491 4408 567328 1507 1526 TTTCTCTTAAGGATTTAATA 5-10-5 MOE 34 10479 10498 4409 567329 1508 1527 CTTTCTCTTAAGGATTTAAT 5-10-5 MOE 46 10480 10499 4410 567330 1509 1528 GCTTTCTCTTAAGGATTTAA 5-10-5 MOE 75 10481 10500 95 567331 1510 1529 AGCTTTCTCTTAAGGATTTA 5-10-5 MOE 59 10482 10501 4411 567332 1511 1530 AAGCTTTCTCTTAAGGATTT 5-10-5 MOE 30 10483 10502 4412 567333 1513 1532 TCAAGCTTTCTCTTAAGGAT 5-10-5 MOE 65 10485 10504 4413 567334 1514 1533 CTCAAGCTTTCTCTTAAGGA 5-10-5 MOE 77 10486 10505 96 567335 1515 1534 TCTCAAGCTTTCTCTTAAGG 5-10-5 MOE 75 10487 10506 97 567336 1516 1535 TTCTCAAGCTTTCTCTTAAG 5-10-5 MOE 59 10488 10507 4414 567337 1517 1536 TTTCTCAAGCTTTCTCTTAA 5-10-5 MOE 52 10489 10508 4415 567338 1521 1540 TCTATTTCTCAAGCTTTCTC 5-10-5 MOE 68 10493 10512 4416 567339 1522 1541 ATCTATTTCTCAAGCTTTCT 5-10-5 MOE 71 10494 10513 4417 567340 1523 1542 AATCTATTTCTCAAGCTTTC 5-10-5 MOE 74 10495 10514 4418 567341 1524 1543 AAATCTATTTCTCAAGCTTT 5-10-5 MOE 63 10496 10515 4419 567342 1525 1544 AAAATCTATTTCTCAAGCTT 5-10-5 MOE 54 10497 10516 4420 567343 1532 1551 GATAAAAAAAATCTATTTCT 5-10-5 MOE 30 10504 10523 4421 567344 1548 1567 TAGACAGTGACTTTAAGATA 5-10-5 MOE 37 10520 10539 4422 567345 1549 1568 ATAGACAGTGACTTTAAGAT 5-10-5 MOE 29 10521 10540 4423 567346 1550 1569 AATAGACAGTGACTTTAAGA 5-10-5 MOE 48 10522 10541 4424 567347 1551 1570 AAATAGACAGTGACTTTAAG 5-10-5 MOE 26 10523 10542 4425 567348 1552 1571 TAAATAGACAGTGACTTTAA 5-10-5 MOE 26 10524 10543 4426 567349 1553 1572 TTAAATAGACAGTGACTTTA 5-10-5 MOE 50 10525 10544 4427 567350 1554 1573 CTTAAATAGACAGTGACTTT 5-10-5 MOE 63 10526 10545 4428 567351 1555 1574 TCTTAAATAGACAGTGACTT 5-10-5 MOE 57 10527 10546 4429 567352 1556 1575 ATCTTAAATAGACAGTGACT 5-10-5 MOE 69 10528 10547 4430 567353 1557 1576 AATCTTAAATAGACAGTGAC 5-10-5 MOE 40 10529 10548 4431 567354 1558 1577 TAATCTTAAATAGACAGTGA 5-10-5 MOE 30 10530 10549 4432 567355 1559 1578 TTAATCTTAAATAGACAGTG 5-10-5 MOE 25 10531 10550 4433 567356 1560 1579 TTTAATCTTAAATAGACAGT 5-10-5 MOE 0 10532 10551 4434 567357 1561 1580 GTTTAATCTTAAATAGACAG 5-10-5 MOE 34 10533 10552 4435 567358 1562 1581 TGTTTAATCTTAAATAGACA 5-10-5 MOE 5 10534 10553 4436 567359 1563 1582 ATGTTTAATCTTAAATAGAC 5-10-5 MOE 0 10535 10554 4437 567360 1567 1586 TTGTATGTTTAATCTTAAAT 5-10-5 MOE 0 10539 10558 4438 567361 1568 1587 ATTGTATGTTTAATCTTAAA 5-10-5 MOE 8 10540 10559 4439 567362 1569 1588 GATTGTATGTTTAATCTTAA 5-10-5 MOE 20 10541 10560 4440 567363 1570 1589 TGATTGTATGTTTAATCTTA 5-10-5 MOE 29 10542 10561 4441 567364 1574 1593 TATGTGATTGTATGTTTAAT 5-10-5 MOE 7 10546 10565 4442 567365 1576 1595 GTTATGTGATTGTATGTTTA 5-10-5 MOE 43 10548 10567 4443 567366 1580 1599 TAAGGTTATGTGATTGTATG 5-10-5 MOE 28 10552 10571 4444 567367 1581 1600 TTAAGGTTATGTGATTGTAT 5-10-5 MOE 31 10553 10572 4445 567368 1585 1604 TTCTTTAAGGTTATGTGATT 5-10-5 MOE 12 10557 10576 4446 561527 1604 1619 GAAATGTAAACGGTAT Deoxy, MOE, and cEt 47 10576 10591 4447 561528 1606 1621 GAGAAATGTAAACGGT Deoxy, MOE, and cEt 89 10578 10593 174 561529 1608 1623 TTGAGAAATGTAAACG Deoxy, MOE, and cEt 55 10580 10595 4448 561530 1611 1626 TGATTGAGAAATGTAA Deoxy, MOE, and cEt 18 10583 10598 4449 561531 1613 1628 TTTGATTGAGAAATGT Deoxy, MOE, and cEt 30 10585 10600 4450 561532 1619 1634 AAGAATTTTGATTGAG Deoxy, MOE, and cEt 53 10591 10606 4451 561533 1621 1636 ATAAGAATTTTGATTG Deoxy, MOE, and cEt 29 10593 10608 4452 561534 1632 1647 CAAATAGTATTATAAG Deoxy, MOE, and cEt 6 10604 10619 4453 561535 1653 1668 CCCACATCACAAAATT Deoxy, MOE, and cEt 70 10625 10640 4454 561536 1657 1672 GATTCCCACATCACAA Deoxy, MOE, and cEt 77 10629 10644 4455 561537 1659 1674 TTGATTCCCACATCAC Deoxy, MOE, and cEt 78 10631 10646 4456 561538 1661 1676 AATTGATTCCCACATC Deoxy, MOE, and cEt 68 10633 10648 4457 561539 1663 1678 AAAATTGATTCCCACA Deoxy, MOE, and cEt 72 10635 10650 4458 561540 1665 1680 CTAAAATTGATTCCCA Deoxy, MOE, and cEt 54 10637 10652 4459 561541 1668 1683 CATCTAAAATTGATTC Deoxy, MOE, and cEt 0 10640 10655 4460 561542 1670 1685 ACCATCTAAAATTGAT Deoxy, MOE, and cEt 35 10642 10657 4461 561543 1672 1687 TGACCATCTAAAATTG Deoxy, MOE, and cEt 55 10644 10659 4462 561544 1674 1689 TGTGACCATCTAAAAT Deoxy, MOE, and cEt 56 10646 10661 4463 561545 1676 1691 ATTGTGACCATCTAAA Deoxy, MOE, and cEt 73 10648 10663 4464 561546 1678 1693 AGATTGTGACCATCTA Deoxy, MOE, and cEt 67 10650 10665 4465 561547 1680 1695 CTAGATTGTGACCATC Deoxy, MOE, and cEt 50 10652 10667 4466 561548 1682 1697 ATCTAGATTGTGACCA Deoxy, MOE, and cEt 77 10654 10669 4467 561549 1684 1699 TAATCTAGATTGTGAC Deoxy, MOE, and cEt 55 10656 10671 4468 561550 1686 1701 TATAATCTAGATTGTG Deoxy, MOE, and cEt 28 10658 10673 4469 561551 1688 1703 ATTATAATCTAGATTG Deoxy, MOE, and cEt 52 10660 10675 4470 561552 1690 1705 TGATTATAATCTAGAT Deoxy, MOE, and cEt 43 10662 10677 4471 561553 1692 1707 ATTGATTATAATCTAG Deoxy, MOE, and cEt 53 10664 10679 4472 561554 1694 1709 CTATTGATTATAATCT Deoxy, MOE, and cEt 54 10666 10681 4473 561555 1696 1711 ACCTATTGATTATAAT Deoxy, MOE, and cEt 44 10668 10683 4474 561556 1698 1713 TCACCTATTGATTATA Deoxy, MOE, and cEt 52 10670 10685 4475 561557 1700 1715 GTTCACCTATTGATTA Deoxy, MOE, and cEt 50 10672 10687 4476 561558 1702 1717 AAGTTCACCTATTGAT Deoxy, MOE, and cEt 58 10674 10689 4477 561559 1704 1719 ATAAGTTCACCTATTG Deoxy, MOE, and cEt 66 10676 10691 4478 561560 1706 1721 TAATAAGTTCACCTAT Deoxy, MOE, and cEt 38 10678 10693 4479 561561 1708 1723 TTTAATAAGTTCACCT Deoxy, MOE, and cEt 50 10680 10695 4480 561562 1710 1725 TATTTAATAAGTTCAC Deoxy, MOE, and cEt 32 10682 10697 4481 561563 1712 1727 GTTATTTAATAAGTTC Deoxy, MOE, and cEt 47 10684 10699 4482 561564 1761 1776 CATATGATGCCTTTTA Deoxy, MOE, and cEt 63 10733 10748 4483 561565 1763 1778 CTCATATGATGCCTTT Deoxy, MOE, and cEt 81 10735 10750 175 561566 1765 1780 AGCTCATATGATGCCT Deoxy, MOE, and cEt 81 10737 10752 176 561567 1767 1782 TTAGCTCATATGATGC Deoxy, MOE, and cEt 84 10739 10754 177 561568 1769 1784 TATTAGCTCATATGAT Deoxy, MOE, and cEt 46 10741 10756 4484 561569 1771 1786 GATATTAGCTCATATG Deoxy, MOE, and cEt 49 10743 10758 4485 561570 1773 1788 GTGATATTAGCTCATA Deoxy, MOE, and cEt 81 10745 10760 4486 561571 1775 1790 TTGTGATATTAGCTCA Deoxy, MOE, and cEt 85 10747 10762 178 561572 1777 1792 AGTTGTGATATTAGCT Deoxy, MOE, and cEt 68 10749 10764 4487 561573 1779 1794 AAAGTTGTGATATTAG Deoxy, MOE, and cEt 45 10751 10766 4488 561574 1781 1796 GGAAAGTTGTGATATT Deoxy, MOE, and cEt 27 10753 10768 4489 561575 1783 1798 TGGGAAAGTTGTGATA Deoxy, MOE, and cEt 36 10755 10770 4490 561576 1785 1800 ACTGGGAAAGTTGTGA Deoxy, MOE, and cEt 83 10757 10772 179 561577 1787 1802 AAACTGGGAAAGTTGT Deoxy, MOE, and cEt 56 10759 10774 4491 561578 1789 1804 TTAAACTGGGAAAGTT Deoxy, MOE, and cEt 44 10761 10776 4492 561579 1794 1809 GTTTTTTAAACTGGGA Deoxy, MOE, and cEt 58 10766 10781 4493 561580 1796 1811 TAGTTTTTTAAACTGG Deoxy, MOE, and cEt 0 10768 10783 4494 561581 1802 1817 GAGTACTAGTTTTTTA Deoxy, MOE, and cEt 18 10774 10789 4495 561582 1804 1819 AAGAGTACTAGTTTTT Deoxy, MOE, and cEt 55 10776 10791 4496 561583 1806 1821 ACAAGAGTACTAGTTT Deoxy, MOE, and cEt 51 10778 10793 4497 561584 1808 1823 TAACAAGAGTACTAGT Deoxy, MOE, and cEt 53 10780 10795 4498 561585 1810 1825 TTTAACAAGAGTACTA Deoxy, MOE, and cEt 48 10782 10797 4499 561586 1812 1827 GTTTTAACAAGAGTAC Deoxy, MOE, and cEt 49 10784 10799 4500 561587 1814 1829 GAGTTTTAACAAGAGT Deoxy, MOE, and cEt 54 10786 10801 4501 561588 1816 1831 TAGAGTTTTAACAAGA Deoxy, MOE, and cEt 9 10788 10803 4502 561589 1819 1834 GTTTAGAGTTTTAACA Deoxy, MOE, and cEt 24 10791 10806 4503 561590 1822 1837 CAAGTTTAGAGTTTTA Deoxy, MOE, and cEt 30 10794 10809 4504 561591 1824 1839 GTCAAGTTTAGAGTTT Deoxy, MOE, and cEt 60 10796 10811 4505 561592 1826 1841 TAGTCAAGTTTAGAGT Deoxy, MOE, and cEt 56 10798 10813 4506 561593 1828 1843 TTTAGTCAAGTTTAGA Deoxy, MOE, and cEt 41 10800 10815 4507 561594 1830 1845 TATTTAGTCAAGTTTA Deoxy, MOE, and cEt 14 10802 10817 4508 561595 1832 1847 TGTATTTAGTCAAGTT Deoxy, MOE, and cEt 39 10804 10819 4509 561596 1834 1849 TCTGTATTTAGTCAAG Deoxy, MOE, and cEt 51 10806 10821 4510 561597 1836 1851 CCTCTGTATTTAGTCA Deoxy, MOE, and cEt 72 10808 10823 4511 561598 1838 1853 GTCCTCTGTATTTAGT Deoxy, MOE, and cEt 55 10810 10825 4512 561599 1840 1855 CAGTCCTCTGTATTTA Deoxy, MOE, and cEt 63 10812 10827 4513 561600 1842 1857 ACCAGTCCTCTGTATT Deoxy, MOE, and cEt 66 10814 10829 4514 561601 1844 1859 TTACCAGTCCTCTGTA Deoxy, MOE, and cEt 57 10816 10831 4515 561602 1846 1861 AATTACCAGTCCTCTG Deoxy, MOE, and cEt 43 10818 10833 4516 561603 1848 1863 ACAATTACCAGTCCTC Deoxy, MOE, and cEt 67 10820 10835 4517

TABLE 154 Inhibition of ANGPTL3 mRNA by oligonucleotides targeting SEQ ID NO: 1 and 2 SEQ SEQ ID SEQ ID SEQ ID NO: 1 NO: ID NO: NO: 2 SEQ ISIS Start 1 Stop % 2 Start Stop ID NO Site Site Sequence Chemistry inhibition Site Site NO 561835 N/A N/A GCAAATTTTCAGTGTT Deoxy, MOE, and cEt 49 3850 3865 4518 561836 N/A N/A CGATTTGTAATTTTCA Deoxy, MOE, and cEt 20 3874 3889 4519 561837 N/A N/A TTTAACCGATTTGTAA Deoxy, MOE, and cEt 42 3880 3895 4520 561838 N/A N/A GTATAATTTAACCGAT Deoxy, MOE, and cEt 15 3886 3901 4521 561839 N/A N/A CTAGATTGTATAATTT Deoxy, MOE, and cEt 15 3893 3908 4522 561840 N/A N/A AGTGTTCTAGATTGTA Deoxy, MOE, and cEt 45 3899 3914 4523 561841 N/A N/A TGACATAGTGTTCTAG Deoxy, MOE, and cEt 51 3905 3920 4524 561842 N/A N/A GTGTAATGACATAGTG Deoxy, MOE, and cEt 58 3911 3926 4525 561843 N/A N/A ACAATAGTGTAATGAC Deoxy, MOE, and cEt 12 3917 3932 4526 561844 N/A N/A GTAATTTACAATAGTG Deoxy, MOE, and cEt 18 3924 3939 4527 561845 N/A N/A CCTTCAGTAATTTACA Deoxy, MOE, and cEt 0 3930 3945 4528 561846 N/A N/A TACTTACCTTCAGTAA Deoxy, MOE, and cEt 2 3936 3951 4529 561847 N/A N/A CTGGAGAATAGTTTTA Deoxy, MOE, and cEt 19 3969 3984 4530 561848 N/A N/A TTAAACACTGGAGAAT Deoxy, MOE, and cEt 14 3976 3991 4531 561849 N/A N/A GCCCAGCATATTTTCA Deoxy, MOE, and cEt 22 4034 4049 4532 561850 N/A N/A GAAAAAGCCCAGCATA Deoxy, MOE, and cEt 15 4040 4055 4533 561851 N/A N/A GATTTTCTGAACTTCA Deoxy, MOE, and cEt 52 4063 4078 4534 561852 N/A N/A GTACTATCTCTAAAAT Deoxy, MOE, and cEt 6 4081 4096 4535 561853 N/A N/A TAAATTGTACTATCTC Deoxy, MOE, and cEt 13 4087 4102 4536 561854 N/A N/A CACATATTTTTGTCCT Deoxy, MOE, and cEt 47 4115 4130 4537 561855 N/A N/A CTTTCAAATAGCACAT Deoxy, MOE, and cEt 31 4126 4141 4538 561856 N/A N/A GTATGCTTCTTTCAAA Deoxy, MOE, and cEt 22 4134 4149 4539 561857 N/A N/A CCCCTTGTATGCTTCT Deoxy, MOE, and cEt 55 4140 4155 4540 561858 N/A N/A TTCCTTCCCCTTGTAT Deoxy, MOE, and cEt 32 4146 4161 4541 561859 N/A N/A TGGCAATTCCTTCCCC Deoxy, MOE, and cEt 43 4152 4167 4542 561860 N/A N/A GAATATTGGCAATTCC Deoxy, MOE, and cEt 52 4158 4173 4543 561861 N/A N/A CTAATAATGGATTTGA Deoxy, MOE, and cEt 0 4179 4194 4544 561862 N/A N/A CTATCATAATCTAAAT Deoxy, MOE, and cEt 0 4202 4217 4545 561863 N/A N/A GTAACACTATCATAAT Deoxy, MOE, and cEt 7 4208 4223 4546 561864 N/A N/A AATTTCCTGTAACACT Deoxy, MOE, and cEt 17 4216 4231 4547 561865 N/A N/A AAGTTGCTTTCCTCTT Deoxy, MOE, and cEt 12 4243 4258 4548 561866 N/A N/A GGTTATAAGTTGCTTT Deoxy, MOE, and cEt 6 4249 4264 4549 561867 N/A N/A TAGGTTGGTTATAAGT Deoxy, MOE, and cEt 10 4255 4270 4550 561868 N/A N/A AGAGAGTAGGTTGGTT Deoxy, MOE, and cEt 10 4261 4276 4551 561869 N/A N/A GGATATAGAGAGTAGG Deoxy, MOE, and cEt 23 4267 4282 4552 561870 N/A N/A AAGTCTGGATATAGAG Deoxy, MOE, and cEt 13 4273 4288 4553 561871 N/A N/A CTACAAAAGTCTGGAT Deoxy, MOE, and cEt 1 4279 4294 4554 561872 N/A N/A CTTACCTGATTTTCTA Deoxy, MOE, and cEt 0 4385 4400 4555 561873 N/A N/A TACTGACTTACCTGAT Deoxy, MOE, and cEt 2 4391 4406 4556 561874 N/A N/A CCATTAAAATACTGAC Deoxy, MOE, and cEt 1 4400 4415 4557 561875 N/A N/A GGACATACCATTAAAA Deoxy, MOE, and cEt 11 4407 4422 4558 561876 N/A N/A AAGATGGGACATACCA Deoxy, MOE, and cEt 38 4413 4428 4559 561877 N/A N/A GTGTGAAAGATGGGAC Deoxy, MOE, and cEt 25 4419 4434 4560 561878 N/A N/A AGACCTGTGTGAAAGA Deoxy, MOE, and cEt 33 4425 4440 4561 561879 N/A N/A TTTTACAGACCTGTGT Deoxy, MOE, and cEt 29 4431 4446 4562 561880 N/A N/A CAGTGTTTTTACAGAC Deoxy, MOE, and cEt 40 4437 4452 4563 561881 N/A N/A TAGGATTCAGTGTTTT Deoxy, MOE, and cEt 62 4444 4459 4564 561882 N/A N/A GTTAAAGCTTGTAAAT Deoxy, MOE, and cEt 16 4465 4480 4565 561883 N/A N/A GATCCAGTTAAAGCTT Deoxy, MOE, and cEt 39 4471 4486 4566 561884 N/A N/A ACTCATGATCCAGTTA Deoxy, MOE, and cEt 60 4477 4492 4567 561885 N/A N/A AATTTTACTCATGATC Deoxy, MOE, and cEt 36 4483 4498 4568 561886 N/A N/A TGTGATAATTTTACTC Deoxy, MOE, and cEt 30 4489 4504 4569 561887 N/A N/A TGCTGATGTGATAATT Deoxy, MOE, and cEt 41 4495 4510 4570 561888 N/A N/A CAGTTATGCTGATGTG Deoxy, MOE, and cEt 86 4501 4516 185 561889 N/A N/A GCAATTTTAACAGTTA Deoxy, MOE, and cEt 13 4511 4526 4571 561890 N/A N/A GAGCCTGCAATTTTAA Deoxy, MOE, and cEt 14 4517 4532 4572 561891 N/A N/A TAGCTTCAGAGCCTGC Deoxy, MOE, and cEt 61 4525 4540 4573 561892 N/A N/A GTTTATTAGCTTCAGA Deoxy, MOE, and cEt 45 4531 4546 4574 561893 N/A N/A CAGGTAGTTTATTAGC Deoxy, MOE, and cEt 37 4537 4552 4575 561894 N/A N/A TAAATGCAGGTAGTTT Deoxy, MOE, and cEt 11 4543 4558 4576 561895 N/A N/A ATGGTTTAAATGCAGG Deoxy, MOE, and cEt 53 4549 4564 4577 561896 N/A N/A TAGAGCCATGGTTTAA Deoxy, MOE, and cEt 58 4556 4571 4578 561897 N/A N/A AAGTTTTAGAGCCATG Deoxy, MOE, and cEt 81 4562 4577 186 561898 N/A N/A TCACACAAAGTTTTAG Deoxy, MOE, and cEt 17 4569 4584 4579 561899 N/A N/A GTGAAGTAATTTATTC Deoxy, MOE, and cEt 8 4589 4604 4580 561900 N/A N/A ACTGAGAGATAAAGGG Deoxy, MOE, and cEt 34 4605 4620 4581 561901 N/A N/A GTATATGTGAGGAAAC Deoxy, MOE, and cEt 18 4619 4634 4582 561902 N/A N/A TTTGTAGTATATGTGA Deoxy, MOE, and cEt 3 4625 4640 4583 561903 N/A N/A ATTATCTTTGTAGTAT Deoxy, MOE, and cEt 8 4631 4646 4584 561904 N/A N/A ATAAGTTCTGTTATTA Deoxy, MOE, and cEt 18 4643 4658 4585 561905 N/A N/A AATCCTATAAGTTCTG Deoxy, MOE, and cEt 55 4649 4664 4586 561906 N/A N/A CTGCTATGAATTAATT Deoxy, MOE, and cEt 16 4679 4694 4587 561907 N/A N/A CATTGGCTGCTATGAA Deoxy, MOE, and cEt 48 4685 4700 4588 561908 N/A N/A AGATGACATTGGCTGC Deoxy, MOE, and cEt 71 4691 4706 4589 561909 N/A N/A TTAGTAAGATGACATT Deoxy, MOE, and cEt 0 4697 4712 4590 561910 N/A N/A GATCTAATTTGAATTT Deoxy, MOE, and cEt 7 4712 4727 4591 561911 N/A N/A TTGAGCAAAGAGAAAC Deoxy, MOE, and cEt 6 4730 4745 4592 561989 N/A N/A GAATGTTGACCTTTAA Deoxy, MOE, and cEt 49 5356 5371 4593 561990 N/A N/A ATTGTTGAATGTTGAC Deoxy, MOE, and cEt 57 5362 5377 4594 561991 N/A N/A TTAATTACATTGTTGA Deoxy, MOE, and cEt 0 5370 5385 4595 561992 N/A N/A TTGTAGATTAATTACA Deoxy, MOE, and cEt 18 5377 5392 4596 561993 N/A N/A TTTACATTGTAGATTA Deoxy, MOE, and cEt 3 5383 5398 4597 561994 N/A N/A CAGATGTTTACATTGT Deoxy, MOE, and cEt 71 5389 5404 4598 561995 N/A N/A CTTCACCAGATGTTTA Deoxy, MOE, and cEt 19 5395 5410 4599 561996 N/A N/A CTGTCACTTCACCAGA Deoxy, MOE, and cEt 77 5401 5416 187 561997 N/A N/A AGTGCTTCCCTCTGTC Deoxy, MOE, and cEt 66 5412 5427 4600 561998 N/A N/A TAAACAAGTGCTTCCC Deoxy, MOE, and cEt 62 5418 5433 4601 561999 N/A N/A TAGCTTTTTTCTAAAC Deoxy, MOE, and cEt 0 5429 5444 4602 562000 N/A N/A CTGACATAGCTTTTTT Deoxy, MOE, and cEt 66 5435 5450 4603 562001 N/A N/A TGGATTCTGACATAGC Deoxy, MOE, and cEt 85 5441 5456 188 562002 N/A N/A AATACATGGATTCTGA Deoxy, MOE, and cEt 35 5447 5462 4604 562003 N/A N/A TATTAGAATACATGGA Deoxy, MOE, and cEt 7 5453 5468 4605 562004 N/A N/A GTACTGCATATTAGAA Deoxy, MOE, and cEt 48 5461 5476 4606 562005 N/A N/A ACTATTGTACTGCATA Deoxy, MOE, and cEt 53 5467 5482 4607 562006 N/A N/A TTTTAAACTATTGTAC Deoxy, MOE, and cEt 0 5473 5488 4608 562007 N/A N/A GAGAGTATTATTAATA Deoxy, MOE, and cEt 8 5490 5505 4609 562008 N/A N/A CTGTTTGAGAGTATTA Deoxy, MOE, and cEt 0 5496 5511 4610 562009 N/A N/A GAATAGCTGTTTGAGA Deoxy, MOE, and cEt 34 5502 5517 4611 562010 N/A N/A AATCCTCTTGAATAGC Deoxy, MOE, and cEt 62 5511 5526 4612 562011 N/A N/A TTTTTGAATCCTCTTG Deoxy, MOE, and cEt 50 5517 5532 4613 562012 N/A N/A GAGTTTATATTATGTT Deoxy, MOE, and cEt 5 5532 5547 4614 562013 N/A N/A GTTTCTCTGAGTTTAT Deoxy, MOE, and cEt 58 5540 5555 4615 562014 N/A N/A TTACCAGTTTCTCTGA Deoxy, MOE, and cEt 64 5546 5561 4616 562015 N/A N/A GATTTTGTTTACCAGT Deoxy, MOE, and cEt 68 5554 5569 4617 562016 N/A N/A GTTTTATATCTCTTGA Deoxy, MOE, and cEt 33 5574 5589 4618 562017 N/A N/A TTGGTAATAATATTTG Deoxy, MOE, and cEt 13 5589 5604 4619 562018 N/A N/A TGGAAATTGGTAATAA Deoxy, MOE, and cEt 1 5595 5610 4620 562019 N/A N/A GTTTAGTGGAAATTGG Deoxy, MOE, and cEt 44 5601 5616 4621 562020 N/A N/A ATGTTTGTTTAGTGGA Deoxy, MOE, and cEt 47 5607 5622 4622 562021 N/A N/A CTAACATTATGTTTGT Deoxy, MOE, and cEt 0 5615 5630 4623 562022 N/A N/A GCACTACTAACATTAT Deoxy, MOE, and cEt 42 5621 5636 4624 562023 N/A N/A TTAGCAGCACTACTAA Deoxy, MOE, and cEt 35 5627 5642 4625 562024 N/A N/A AACCTTTTAGCAGCAC Deoxy, MOE, and cEt 76 5633 5648 189 562025 N/A N/A TTGATAAAAAACCTTT Deoxy, MOE, and cEt 0 5642 5657 4626 562026 N/A N/A CAAAAGTAGTTGATAA Deoxy, MOE, and cEt 0 5651 5666 4627 562027 N/A N/A GGAAACCAAAAGTAGT Deoxy, MOE, and cEt 28 5657 5672 4628 562028 N/A N/A GAAAGTATGGAAACCA Deoxy, MOE, and cEt 52 5665 5680 4629 562029 N/A N/A ACATCATAAGAAGGAA Deoxy, MOE, and cEt 8 5678 5693 4630 562030 N/A N/A TCATAGTAAAAGATAT Deoxy, MOE, and cEt 0 5718 5733 4631 562031 N/A N/A TCATTTAATCATAGTA Deoxy, MOE, and cEt 7 5726 5741 4632 562032 N/A N/A GCAGGTTCATTTAATC Deoxy, MOE, and cEt 56 5732 5747 4633 562033 N/A N/A GTAACATTTTGCTTTG Deoxy, MOE, and cEt 44 5752 5767 4634 562034 N/A N/A ATATTACTATAGTAAC Deoxy, MOE, and cEt 4 5763 5778 4635 562035 N/A N/A CAATGTATATTACTAT Deoxy, MOE, and cEt 19 5769 5784 4636 562036 N/A N/A TAGACACAATGTATAT Deoxy, MOE, and cEt 17 5775 5790 4637 562037 N/A N/A GGTTTCTTCACACATT Deoxy, MOE, and cEt 63 5799 5814 4638 562038 N/A N/A CTCAGAAATTCATTGT Deoxy, MOE, and cEt 36 5818 5833 4639 562039 N/A N/A CTTCTTCCAACTCAGA Deoxy, MOE, and cEt 56 5828 5843 4640 562040 N/A N/A CTAACTCTTCTTCCAA Deoxy, MOE, and cEt 39 5834 5849 4641 562041 N/A N/A AATGATCTAACTCTTC Deoxy, MOE, and cEt 33 5840 5855 4642 562042 N/A N/A GAAAGTTAAATGATCT Deoxy, MOE, and cEt 3 5848 5863 4643 562043 N/A N/A ATCTTAAAGTTACTTA Deoxy, MOE, and cEt 56 5900 5915 4644 562044 N/A N/A TATGTGATCTTAAAGT Deoxy, MOE, and cEt 5 5906 5921 4645 562045 N/A N/A AGTAACTATGTGATCT Deoxy, MOE, and cEt 60 5912 5927 4646 562046 N/A N/A CTACTAAGTAACTATG Deoxy, MOE, and cEt 0 5918 5933 4647 562047 N/A N/A TCTTTTCTACTAAGTA Deoxy, MOE, and cEt 18 5924 5939 4648 562048 N/A N/A TATTACTCTTTTCTAC Deoxy, MOE, and cEt 3 5930 5945 4649 562049 N/A N/A GCTGGGTATTACTCTT Deoxy, MOE, and cEt 76 5936 5951 4650 562050 N/A N/A TTGCTTGCTGGGTATT Deoxy, MOE, and cEt 77 5942 5957 190 562051 N/A N/A TAAAGTTTGCTTGCTG Deoxy, MOE, and cEt 58 5948 5963 4651 562052 N/A N/A CTATTGTAAAGTTTGC Deoxy, MOE, and cEt 16 5954 5969 4652 562053 N/A N/A AAGGATCTATTGTAAA Deoxy, MOE, and cEt 5 5960 5975 4653 562054 N/A N/A CTTATTTAAAAGGATC Deoxy, MOE, and cEt 0 5969 5984 4654 562055 N/A N/A TAGGACCTTATTTAAA Deoxy, MOE, and cEt 0 5975 5990 4655 562056 N/A N/A ATTTCCTAGGACCTTA Deoxy, MOE, and cEt 10 5981 5996 4656 562057 N/A N/A CATGAATGATATTTCC Deoxy, MOE, and cEt 39 5991 6006 4657 562058 N/A N/A TGCTGGCATGAATGAT Deoxy, MOE, and cEt 62 5997 6012 4658 562059 N/A N/A TTTTGATGCTGGCATG Deoxy, MOE, and cEt 74 6003 6018 4659 562060 N/A N/A TTAGTTTTTTGATGCT Deoxy, MOE, and cEt 25 6009 6024 4660 562061 N/A N/A GCATTATTAGTGTTAG Deoxy, MOE, and cEt 44 6021 6036 4661 562062 N/A N/A TATCTTGCATTATTAG Deoxy, MOE, and cEt 35 6027 6042 4662 562063 N/A N/A ATATAATATCTTGCAT Deoxy, MOE, and cEt 0 6033 6048 4663 562064 N/A N/A CATTGACAGTAAGAAA Deoxy, MOE, and cEt 0 6057 6072 4664 562065 N/A N/A AGTTTTTCTCATTGAC Deoxy, MOE, and cEt 62 6066 6081 4665 562143 N/A N/A ATGGATATCTCTTAAC Deoxy, MOE, and cEt 18 6869 6884 4666 562144 N/A N/A TATTTGATGGATATCT Deoxy, MOE, and cEt 35 6875 6890 4667 562145 N/A N/A ACATTGTATTTGATGG Deoxy, MOE, and cEt 41 6881 6896 4668 562146 N/A N/A GTTGATACATTGTATT Deoxy, MOE, and cEt 8 6887 6902 4669 562147 N/A N/A GTTTAGGTTGATACAT Deoxy, MOE, and cEt 35 6893 6908 4670 562148 N/A N/A CATCCAGTTTAGGTTG Deoxy, MOE, and cEt 59 6899 6914 4671 562149 N/A N/A CCCCAGCATCCAGTTT Deoxy, MOE, and cEt 37 6905 6920 4672 562150 N/A N/A AAAGAACCCCAGCATC Deoxy, MOE, and cEt 35 6911 6926 4673 562151 N/A N/A GTGTAAAAAGAACCCC Deoxy, MOE, and cEt 33 6917 6932 4674 562152 N/A N/A TATAGGGTGTAAAAAG Deoxy, MOE, and cEt 0 6923 6938 4675 562153 N/A N/A GTCTTTTATAGGGTGT Deoxy, MOE, and cEt 75 6929 6944 191 562154 N/A N/A AGGTATGTCTTTTATA Deoxy, MOE, and cEt 21 6935 6950 4676 562155 N/A N/A TTGTCTTAGGTATGTC Deoxy, MOE, and cEt 84 6942 6957 192 562156 N/A N/A CTCTGATTGTCTTAGG Deoxy, MOE, and cEt 77 6948 6963 193 562157 N/A N/A GTATTTCTCTGATTGT Deoxy, MOE, and cEt 77 6954 6969 194 562158 N/A N/A AGTCCATATTTGTATT Deoxy, MOE, and cEt 49 6965 6980 4677 562159 N/A N/A TAATCAAGTCCATATT Deoxy, MOE, and cEt 19 6971 6986 4678 562160 N/A N/A ATCTAATAATCAAGTC Deoxy, MOE, and cEt 5 6977 6992 4679 562161 N/A N/A CCTTCTATATTATCTA Deoxy, MOE, and cEt 38 6988 7003 4680 562162 N/A N/A TAATAAACCTTCTATA Deoxy, MOE, and cEt 8 6995 7010 4681 562163 N/A N/A GATCACATCTAAGAAA Deoxy, MOE, and cEt 25 7013 7028 4682 562164 N/A N/A TACCATGATCACATCT Deoxy, MOE, and cEt 66 7019 7034 4683 562165 N/A N/A CTGCAATACCATGATC Deoxy, MOE, and cEt 54 7025 7040 4684 562166 N/A N/A GTTCTCCTTTAAAACT Deoxy, MOE, and cEt 0 7039 7054 4685 562167 N/A N/A GAGATTGTTCTCCTTT Deoxy, MOE, and cEt 7 7045 7060 4686 562168 N/A N/A AAACAGGAGATTGTTC Deoxy, MOE, and cEt 6 7051 7066 4687 562169 N/A N/A TCTCTTAAACAGGAGA Deoxy, MOE, and cEt 1 7057 7072 4688 562170 N/A N/A CATGTATCTCTTAAAC Deoxy, MOE, and cEt 40 7063 7078 4689 562171 N/A N/A CGTAAATATTTCAGCA Deoxy, MOE, and cEt 30 7077 7092 4690 562172 N/A N/A TAACTCCGTAAATATT Deoxy, MOE, and cEt 0 7083 7098 4691 562173 N/A N/A GACCTTTAACTCCGTA Deoxy, MOE, and cEt 68 7089 7104 4692 562174 N/A N/A TCCAGTGACCTTTAAC Deoxy, MOE, and cEt 6 7095 7110 4693 562175 N/A N/A CACCAGTCTGGAGTCC Deoxy, MOE, and cEt 52 7108 7123 4694 562176 N/A N/A TTCTATCACCAGTCTG Deoxy, MOE, and cEt 67 7114 7129 4695 562177 N/A N/A ATCTTACCAAACTATT Deoxy, MOE, and cEt 23 7171 7186 4696 562178 N/A N/A AGAATCATCTTACCAA Deoxy, MOE, and cEt 55 7177 7192 4697 562179 N/A N/A GAATGTAAGAATCATC Deoxy, MOE, and cEt 0 7184 7199 4698 562180 N/A N/A GTGTTATTTAAGAATG Deoxy, MOE, and cEt 0 7195 7210 4699 562181 N/A N/A TTTTTCTTAGATGGCG Deoxy, MOE, and cEt 82 7210 7225 195 562182 N/A N/A GTTTATGTTAAAGCAT Deoxy, MOE, and cEt 8 7225 7240 4700 562183 N/A N/A AGTAATGTTTATGTTA Deoxy, MOE, and cEt 4 7231 7246 4701 562184 N/A N/A GTAGCATTTTTTCAGT Deoxy, MOE, and cEt 58 7244 7259 4702 562185 N/A N/A GCAAATGTAGCATTTT Deoxy, MOE, and cEt 61 7250 7265 4703 562186 N/A N/A GTTGTGGCAAATGTAG Deoxy, MOE, and cEt 32 7256 7271 4704 562187 N/A N/A TATGAAGTTGTGGCAA Deoxy, MOE, and cEt 54 7262 7277 4705 562188 N/A N/A GATTTCACTTGACATT Deoxy, MOE, and cEt 19 7279 7294 4706 562189 N/A N/A GCTTGAGATTTCACTT Deoxy, MOE, and cEt 42 7285 7300 4707 562190 N/A N/A TTTGGAGCTTGAGATT Deoxy, MOE, and cEt 22 7291 7306 4708 562191 N/A N/A AATATCTTTGGAGCTT Deoxy, MOE, and cEt 36 7297 7312 4709 562192 N/A N/A AGGAATAATATCTTTG Deoxy, MOE, and cEt 5 7303 7318 4710 562193 N/A N/A ATTTAGTAATAGGAAT Deoxy, MOE, and cEt 5 7313 7328 4711 562194 N/A N/A CATCAGATTTAGTAAT Deoxy, MOE, and cEt 0 7319 7334 4712 562195 N/A N/A GTTATTACATCAGATT Deoxy, MOE, and cEt 23 7326 7341 4713 562196 N/A N/A GCCTAGAATCAATAAA Deoxy, MOE, and cEt 8 7344 7359 4714 562197 N/A N/A AGGAATGCCTAGAATC Deoxy, MOE, and cEt 2 7350 7365 4715 562198 N/A N/A TTCAGCAGGAATGCCT Deoxy, MOE, and cEt 46 7356 7371 4716 562199 N/A N/A TTACCTGATATAACAT Deoxy, MOE, and cEt 41 7460 7475 4717 562200 N/A N/A CAGGTTTTACCTGATA Deoxy, MOE, and cEt 31 7466 7481 4718 562201 N/A N/A CTTAGACAGGTTTTAC Deoxy, MOE, and cEt 41 7472 7487 4719 562202 N/A N/A ATTCTCCTTAGACAGG Deoxy, MOE, and cEt 37 7478 7493 4720 562203 N/A N/A CTGTCTATTCTCCTTA Deoxy, MOE, and cEt 53 7484 7499 4721 562204 N/A N/A TAACTACTGTCTATTC Deoxy, MOE, and cEt 5 7490 7505 4722 562205 N/A N/A TTGAACTAACTACTGT Deoxy, MOE, and cEt 3 7496 7511 4723 562206 N/A N/A AGTAAGTTGAACTAAC Deoxy, MOE, and cEt 11 7502 7517 4724 562207 N/A N/A GTAATGAGTAAGTTGA Deoxy, MOE, and cEt 37 7508 7523 4725 562208 N/A N/A TAATCTTCCTAATACG Deoxy, MOE, and cEt 5 7523 7538 4726 562209 N/A N/A ACCAGGTTAATCTTCC Deoxy, MOE, and cEt 71 7530 7545 4727 562210 N/A N/A ATGATAACCAGGTTAA Deoxy, MOE, and cEt 42 7536 7551 4728 562211 N/A N/A CGAATACTCATATATA Deoxy, MOE, and cEt 20 7576 7591 4729 562212 N/A N/A TTTATACGAATACTCA Deoxy, MOE, and cEt 17 7582 7597 4730 562213 N/A N/A ATTATATTTATACGAA Deoxy, MOE, and cEt 0 7588 7603 4731 562214 N/A N/A GGTAAAAGTATTATAT Deoxy, MOE, and cEt 0 7597 7612 4732 562215 N/A N/A GAGAATATTGAGTAAA Deoxy, MOE, and cEt 9 7624 7639 4733 562216 N/A N/A CAGATTATTTTAGAGG Deoxy, MOE, and cEt 16 7645 7660 4734 562217 N/A N/A TCACTTCAGATTATTT Deoxy, MOE, and cEt 34 7651 7666 4735 562218 N/A N/A TAATAGTCACTTCAGA Deoxy, MOE, and cEt 33 7657 7672 4736 562219 N/A N/A TATTGATAATAGTCAC Deoxy, MOE, and cEt 1 7663 7678 4737 562297 N/A N/A TACTATTTGTAATCAA Deoxy, MOE, and cEt 0 8493 8508 4738 562298 N/A N/A CTTGCTTATTTTACTA Deoxy, MOE, and cEt 24 8504 8519 4739 562299 N/A N/A CATCTGTTATTTTATC Deoxy, MOE, and cEt 0 8519 8534 4740 562300 N/A N/A ATGTGCTTTTTGGATT Deoxy, MOE, and cEt 20 8540 8555 4741 562301 N/A N/A GGATTTTTGTATGTGC Deoxy, MOE, and cEt 64 8550 8565 4742 562302 N/A N/A CATCATTCATGGATTT Deoxy, MOE, and cEt 55 8560 8575 4743 562303 N/A N/A CTTAGACATCATTCAT Deoxy, MOE, and cEt 32 8566 8581 4744 562304 N/A N/A TGAGTACTTAGACATC Deoxy, MOE, and cEt 58 8572 8587 4745 562305 N/A N/A TATAAGTGAGTACTTA Deoxy, MOE, and cEt 3 8578 8593 4746 562306 N/A N/A CTACTTTATAAGTGAG Deoxy, MOE, and cEt 0 8584 8599 4747 562307 N/A N/A TGAATGTCTTCTACTT Deoxy, MOE, and cEt 42 8594 8609 4748 562308 N/A N/A TATAATAATGAATGTC Deoxy, MOE, and cEt 2 8602 8617 4749 562309 N/A N/A GTACTGAGCATTTAAA Deoxy, MOE, and cEt 24 8625 8640 4750 562310 N/A N/A CAAATAGTACTGAGCA Deoxy, MOE, and cEt 48 8631 8646 4751 562311 N/A N/A AATGGTCAAATAGTAC Deoxy, MOE, and cEt 0 8637 8652 4752 562312 N/A N/A GTAGTTTGAATACAAA Deoxy, MOE, and cEt 9 8660 8675 4753 562313 N/A N/A TCACTGGTAGTTTGAA Deoxy, MOE, and cEt 56 8666 8681 4754 562314 N/A N/A GGGCTTTCACTGGTAG Deoxy, MOE, and cEt 70 8672 8687 196 562315 N/A N/A TAGGTAGGGCTTTCAC Deoxy, MOE, and cEt 50 8678 8693 4755 562316 N/A N/A ACCTTCTAGGTAGGGC Deoxy, MOE, and cEt 47 8684 8699 4756 562317 N/A N/A GAGTATACCTTCTAGG Deoxy, MOE, and cEt 38 8690 8705 4757 562318 N/A N/A ATCACTGAGTATACCT Deoxy, MOE, and cEt 61 8696 8711 4758 562319 N/A N/A AAACTTATCACTGAGT Deoxy, MOE, and cEt 0 8702 8717 4759 562320 N/A N/A GCTACAAAACTTATCA Deoxy, MOE, and cEt 8 8708 8723 4760 562321 N/A N/A TTTGGAGCTACAAAAC Deoxy, MOE, and cEt 0 8714 8729 4761 562322 N/A N/A AGAAGATTTGGAGCTA Deoxy, MOE, and cEt 24 8720 8735 4762 562323 N/A N/A ACTATTAGAAGATTTG Deoxy, MOE, and cEt 0 8726 8741 4763 562324 N/A N/A ACACTCACTATTAGAA Deoxy, MOE, and cEt 0 8732 8747 4764 562325 N/A N/A AGCCTTTTATTTTGGG Deoxy, MOE, and cEt 37 8751 8766 4765 562326 N/A N/A CCTGTCAGCCTTTTAT Deoxy, MOE, and cEt 0 8757 8772 4766 562327 N/A N/A GACTTACCTGTCAGCC Deoxy, MOE, and cEt 47 8763 8778 4767 562328 N/A N/A ATTCTCGACTTACCTG Deoxy, MOE, and cEt 12 8769 8784 4768 562329 N/A N/A GTGAGTATTCTCGACT Deoxy, MOE, and cEt 25 8775 8790 4769 562330 N/A N/A AATTAAGTGAGTATTC Deoxy, MOE, and cEt 0 8781 8796 4770 562331 N/A N/A TACCAGAATTAAGTGA Deoxy, MOE, and cEt 0 8787 8802 4771 562332 N/A N/A GCTTTCTTACCAGAAT Deoxy, MOE, and cEt 23 8794 8809 4772 562333 N/A N/A TGGGTTGCTTTCTTAC Deoxy, MOE, and cEt 0 8800 8815 4773 562334 N/A N/A TACAAGTACAAATGGG Deoxy, MOE, and cEt 36 8812 8827 4774 562335 N/A N/A GGTAAATACAAGTACA Deoxy, MOE, and cEt 19 8818 8833 4775 562336 N/A N/A ATTGCTGGTAAATACA Deoxy, MOE, and cEt 13 8824 8839 4776 562337 N/A N/A TTAAGGATTGCTGGTA Deoxy, MOE, and cEt 43 8830 8845 4777 562338 N/A N/A GCTTCATTTTAAGGAT Deoxy, MOE, and cEt 12 8838 8853 4778 562339 N/A N/A GTAGGAAGCTTCATTT Deoxy, MOE, and cEt 23 8845 8860 4779 562340 N/A N/A GAGTTAGTAGGAAGCT Deoxy, MOE, and cEt 58 8851 8866 4780 562341 N/A N/A GCTATTGAGTTAGTAG Deoxy, MOE, and cEt 21 8857 8872 4781 562342 N/A N/A CTTATTGCTATTGAGT Deoxy, MOE, and cEt 34 8863 8878 4782 562343 N/A N/A TATTGTCTTATTGCTA Deoxy, MOE, and cEt 17 8869 8884 4783 562344 N/A N/A ATTCACTATTGTCTTA Deoxy, MOE, and cEt 22 8875 8890 4784 562345 N/A N/A ATCACAATCCTTTTTA Deoxy, MOE, and cEt 18 8925 8940 4785 562346 N/A N/A TTCTTCATCACAATCC Deoxy, MOE, and cEt 43 8931 8946 4786 562347 N/A N/A AGATTGTTCTTCATCA Deoxy, MOE, and cEt 35 8937 8952 4787 562348 N/A N/A TATAAATAGATTGTTC Deoxy, MOE, and cEt 10 8944 8959 4788 562349 N/A N/A GGTTCTTAATAACTTT Deoxy, MOE, and cEt 31 9011 9026 4789 562350 N/A N/A AAGCATGGTTCTTAAT Deoxy, MOE, and cEt 12 9017 9032 4790 562351 N/A N/A CTTTGTAGAAAAAGAC Deoxy, MOE, and cEt 0 9066 9081 4791 562352 N/A N/A TATGCTTTCTTTGTAG Deoxy, MOE, and cEt 26 9074 9089 4792 562353 N/A N/A CTTAATGTATGCTTTC Deoxy, MOE, and cEt 55 9081 9096 4793 562354 N/A N/A GTATTTGCTTAATGTA Deoxy, MOE, and cEt 0 9088 9103 4794 562355 N/A N/A CCTTTGGTATTTGCTT Deoxy, MOE, and cEt 54 9094 9109 4795 562356 N/A N/A ACCTGGCCTTTGGTAT Deoxy, MOE, and cEt 0 9100 9115 4796 562357 N/A N/A ATGTAAACCTGGCCTT Deoxy, MOE, and cEt 1 9106 9121 4797 562358 N/A N/A CTTCAAATGTAAACCT Deoxy, MOE, and cEt 0 9112 9127 4798 562359 N/A N/A GTAATAATAATGTCAC Deoxy, MOE, and cEt 0 9131 9146 4799 562360 N/A N/A AGACTTGAGTAATAAT Deoxy, MOE, and cEt 0 9139 9154 4800 562361 N/A N/A TCCTAGAGACTTGAGT Deoxy, MOE, and cEt 25 9145 9160 4801 562362 N/A N/A AAGTATTCCTAGAGAC Deoxy, MOE, and cEt 28 9151 9166 4802 562363 N/A N/A TGTGTTAAGTATTCCT Deoxy, MOE, and cEt 50 9157 9172 4803 562364 N/A N/A AAGAGATGTGTTAAGT Deoxy, MOE, and cEt 21 9163 9178 4804 562365 N/A N/A ACAGTCAAGAGATGTG Deoxy, MOE, and cEt 74 9169 9184 197 562366 N/A N/A CCATATACAGTCAAGA Deoxy, MOE, and cEt 49 9175 9190 4805 562367 N/A N/A TAACATCCATATACAG Deoxy, MOE, and cEt 16 9181 9196 4806 562368 N/A N/A CTATTTATTAACATCC Deoxy, MOE, and cEt 2 9189 9204 4807 562369 N/A N/A TGTCAGCTATTTATTA Deoxy, MOE, and cEt 22 9195 9210 4808 562370 N/A N/A CTTTACTGTCAGCTAT Deoxy, MOE, and cEt 56 9201 9216 4809 562371 N/A N/A GATAAACTTTACTGTC Deoxy, MOE, and cEt 37 9207 9222 4810 562372 N/A N/A CTTTATATGGATAAAC Deoxy, MOE, and cEt 31 9216 9231 4811 562373 N/A N/A GCAAGTCTTTATATGG Deoxy, MOE, and cEt 62 9222 9237 4812 560990 709 724 TTCTTGGTGCTCTTGG Deoxy, MOE, and cEt 74 6722 6737 111 337487 804 823 CACTTGTATGTTCACCTCTG 5-10-5 MOE 30 7389 7408 28 233717 889 908 TGAATTAATGTCCATGGACT 5-10-5 MOE 38 7876 7895 14

Example 121: Antisense Inhibition of Human ANGPTL3 in Hep3B Cells by MOE Gapmers

Additional antisense oligonucleotides were designed targeting an ANGPTL3 nucleic acid and were tested for their effects on ANGPTL3 mRNA in vitro. Cultured Hep3B cells at a density of 20,000 cells per well were transfected using electroporation with 4,500 nM antisense oligonucleotide. After a treatment period of approximately 24 hours, RNA was isolated from the cells and ANGPTL3 mRNA levels were measured by quantitative real-time PCR. Human primer probe set RTS3492_MGB was used to measure mRNA levels. ANGPTL3 mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of ANGPTL3, relative to untreated control cells.

The newly designed chimeric antisense oligonucleotides in the Tables below were designed as 5-10-5 MOE or 3-10-4 MOE gapmers. The 5-10-5 MOE gapmers are 20 nucleosides in length, wherein the central gap segment comprises often 2′-deoxynucleosides and is flanked by wing segments on the 5′ direction and the 3′ direction comprising five nucleosides each. The 3-10-4 MOE gapmers are 17 nucleosides in length, wherein the central gap segment comprises often 2′-deoxynucleosides and is flanked by wing segments on the 5′ direction and the 3′ direction comprising three and four nucleosides respectively. Each nucleoside in the 5′ wing segment and each nucleoside in the 3′ wing segment has a 2′-MOE modification. Each nucleoside in the 5′ wing segment and each nucleoside in the 3′ wing segment has a 2′-MOE modification. The internucleoside linkages throughout each gapmer are phosphorothioate (P═S) linkages. All cytosine residues throughout each gapmer are 5-methylcytosines. “Start site” indicates the 5′-most nucleoside to which the gapmer is targeted in the human gene sequence. “Stop site” indicates the 3′-most nucleoside to which the gapmer is targeted human gene sequence. Each gapmer listed in the Tables below is targeted to either the human ANGPTL3 mRNA, designated herein as SEQ ID NO: 1 (GENBANK Accession No. NM_014495.2) or the human ANGPTL3 genomic sequence, designated herein as SEQ ID NO: 2 (GENBANK Accession No. NT_032977.9 truncated from nucleotides 33032001 to 33046000). ‘n/a’ indicates that the antisense oligonucleotide does not target that particular gene sequence with 100% complementarity.

TABLE 155 Inhibition of ANGPTL3 mRNA by MOE gapmers targeting SEQ ID NO: 1 and 2 SEQ SEQ SEQ SEQ ID NO: ID NO: ID NO: ID NO: ISIS 1 Start 1 Stop % 2 Start 2 Stop SEQ NO Site Site Sequence Motif inhibition Site Site ID NO 582715 N/A N/A CTGGGTATTACTCTTTTCTA 5-10-5 60 5931 5950 4813 582716 N/A N/A CTTGCTGGGTATTACTCTTT 5-10-5 59 5935 5954 4814 582717 N/A N/A TGCTTGCTGGGTATTACTCT 5-10-5 59 5937 5956 4815 582718 N/A N/A CATGAATGATATTTCCTAGG 5-10-5 39 5987 6006 4816 582719 N/A N/A GGCATGAATGATATTTCCTA 5-10-5 60 5989 6008 4817 582720 N/A N/A CTGGCATGAATGATATTTCC 5-10-5 46 5991 6010 4818 582721 N/A N/A TGCTGGCATGAATGATATTT 5-10-5 32 5993 6012 4819 582722 N/A N/A AAGTCCATATTTGTATTTCT 5-10-5 50 6962 6981 4820 582723 N/A N/A GCAAATGTAGCATTTTTTCA 5-10-5 32 7246 7265 4821 582724 N/A N/A GGCAAATGTAGCATTTTTTC 5-10-5 55 7247 7266 4822 582725 N/A N/A GTGGCAAATGTAGCATTTTT 5-10-5 62 7249 7268 203 582726 N/A N/A CTGGTCCTTTTAACTTCCAA 5-10-5 40 8366 8385 4823 582727 N/A N/A CCTGGTCCTTTTAACTTCCA 5-10-5 58 8367 8386 4824 582728 N/A N/A TTCCTGGTCCTTTTAACTTC 5-10-5 32 8369 8388 4825 582729 N/A N/A TGCTTAATGTATGCTTTCTT 5-10-5 51 9079 9098 4826 582730 N/A N/A CCGTAAGTTTATCTTCCTTT 5-10-5 58 10136 10155 4827 582731 N/A N/A CCCCGTAAGTTTATCTTCCT 5-10-5 51 10138 10157 4828 582732 N/A N/A CACAAATATGTTCATTCTTA 5-10-5 22 11189 11208 4829 582733 N/A N/A GCCACAAATATGTTCATTCT 5-10-5 71 11191 11210 204 582734 N/A N/A AAACTTTAACTCGATGCCAC 5-10-5 51 11206 11225 4830 582735 N/A N/A ATAAACTTTAACTCGATGCC 5-10-5 57 11208 11227 4831 582736 N/A N/A ATGCTTGTCAGGCTGTTTAA 5-10-5 56 11311 11330 4832 582737 N/A N/A GTCACCATATAACTTGGGCA 5-10-5 48 11562 11581 4833 582738 N/A N/A AGGTCACCATATAACTTGGG 5-10-5 44 11564 11583 4834 582766 N/A N/A GCTGGGTATTACTCTTT 3-10-4 55 5935 5951 4835 582767 N/A N/A GCATGAATGATATTTCC 3-10-4 4 5991 6007 4836 582768 N/A N/A GGCAAATGTAGCATTTT 3-10-4 33 7250 7266 4837 582769 N/A N/A CTGGTCCTTTTAACTTC 3-10-4 29 8369 8385 4838 582770 N/A N/A GTAAGTTTATCTTCCTT 3-10-4 26 10137 10153 4839 582771 N/A N/A ACTTTAACTCGATGCCA 3-10-4 42 11207 11223 4840 582772 N/A N/A AACTTTAACTCGATGCC 3-10-4 55 11208 11224 4841 582773 N/A N/A AAACTTTAACTCGATGC 3-10-4 1 11209 11225 4842 582774 N/A N/A GCTTGTCAGGCTGTTTA 3-10-4 65 11312 11328 208 582775 N/A N/A CACCATATAACTTGGGC 3-10-4 38 11563 11579 4843 582776 N/A N/A TCACCATATAACTTGGG 3-10-4 37 11564 11580 4844 582777 N/A N/A GTCACCATATAACTTGG 3-10-4 31 11565 11581 4845 582702 139 158 CTTGATTTTGGCTCTGGAGA 5-10-5 53 3243 3262 4846 582739 140 156 TGATTTTGGCTCTGGAG 3-10-4 41 3244 3260 4847 582703 141 160 ATCTTGATTTTGGCTCTGGA 5-10-5 64 3245 3264 198 582740 305 321 ACTGGTTTGCAGCGATA 3-10-4 58 3409 3425 4848 582704 306 325 TTTCACTGGTTTGCAGCGAT 5-10-5 60 3410 3429 4849 582741 306 322 CACTGGTTTGCAGCGAT 3-10-4 57 3410 3426 4850 582742 307 323 TCACTGGTTTGCAGCGA 3-10-4 60 3411 3427 4851 582705 706 725 GTTCTTGGTGCTCTTGGCTT 5-10-5 78 6719 6738 199 544120 707 726 AGTTCTTGGTGCTCTTGGCT 5-10-5 75 6720 6739 15 582743 707 723 TCTTGGTGCTCTTGGCT 3-10-4 63 6720 6736 205 582706 708 727 TAGTTCTTGGTGCTCTTGGC 5-10-5 69 6721 6740 200 582744 708 724 TTCTTGGTGCTCTTGGC 3-10-4 51 6721 6737 4852 582745 709 725 GTTCTTGGTGCTCTTGG 3-10-4 50 6722 6738 4853 337487 804 823 CACTTGTATGTTCACCTCTG 5-10-5 25 7389 7408 28 233717 889 908 TGAATTAATGTCCATGGACT 5-10-5 22 7876 7895 14 582707 1054 1073 TTGTCTTTCCAGTCTTCCAA 5-10-5 42 9629 9648 4854 582708 1056 1075 TGTTGTCTTTCCAGTCTTCC 5-10-5 52 9631 9650 4855 582746 1140 1156 CATTGCCAGTAATCGCA 3-10-4 53 9715 9731 4856 582747 1141 1157 ACATTGCCAGTAATCGC 3-10-4 61 9716 9732 4857 582748 1142 1158 GACATTGCCAGTAATCG 3-10-4 34 9717 9733 4858 582709 1194 1213 CTTTGTGATCCCAAGTAGAA 5-10-5 28 9769 9788 4859 582749 1195 1211 TTGTGATCCCAAGTAGA 3-10-4 16 9770 9786 4860 582710 1196 1215 TGCTTTGTGATCCCAAGTAG 5-10-5 54 9771 9790 4861 582750 1196 1212 TTTGTGATCCCAAGTAG 3-10-4 19 9771 9787 4862 582751 1197 1213 CTTTGTGATCCCAAGTA 3-10-4 32 9772 9788 4863 582752 1260 1276 CACACTCATCATGCCAC 3-10-4 42 10232 10248 4864 582711 1268 1287 GTTGTTTTCTCCACACTCAT 5-10-5 51 10240 10259 4865 582712 1270 1289 AGGTTGTTTTCTCCACACTC 5-10-5 63 10242 10261 201 582753 1307 1323 AGATTTTGCTCTTGGTT 3-10-4 54 10279 10295 4866 582754 1308 1324 TAGATTTTGCTCTTGGT 3-10-4 52 10280 10296 4867 582755 1309 1325 TTAGATTTTGCTCTTGG 3-10-4 44 10281 10297 4868 582756 1310 1326 CTTAGATTTTGCTCTTG 3-10-4 34 10282 10298 4869 567320 1487 1506 CCAGATTATTAGACCACATT 5-10-5 77 10459 10478 93 582757 1488 1504 AGATTATTAGACCACAT 3-10-4 0 10460 10476 4870 582758 1489 1505 CAGATTATTAGACCACA 3-10-4 39 10461 10477 4871 582759 1490 1506 CCAGATTATTAGACCAC 3-10-4 63 10462 10478 206 582760 1491 1507 ACCAGATTATTAGACCA 3-10-4 31 10463 10479 4872 582761 1763 1779 GCTCATATGATGCCTTT 3-10-4 71 10735 10751 207 582713 1906 1925 ACACATACTCTGTGCTGACG 5-10-5 68 10878 10897 202 582762 1907 1923 ACATACTCTGTGCTGAC 3-10-4 57 10879 10895 4873 582714 1908 1927 TTACACATACTCTGTGCTGA 5-10-5 49 10880 10899 4874 582763 2071 2087 CTTAGTAGTCATCTCCA 3-10-4 49 11043 11059 4875 582764 2072 2088 ACTTAGTAGTCATCTCC 3-10-4 53 11044 11060 4876 582765 2073 2089 GACTTAGTAGTCATCTC 3-10-4 36 11045 11061 4877

Example 122: Dose-Dependent Antisense Inhibition of Human ANGPTL3 in Hep3B Cells

Deoxy, MOE, and cEt oligonucleotides from the studies described above exhibiting significant in vitro inhibition of ANGPTL3 mRNA were selected and tested at various doses in Hep3B cells. ISIS 233717 and ISIS 337847, both 5-10-5 MOE gapmers, were also included in the studies. The antisense oligonucleotides were tested in a series of experiments that had similar culture conditions. The results of each experiment are presented in separate tables below.

Cells were plated at a density of 20,000 cells per well and transfected using electroporation with 0.813 μM, 1.625 μM, 3.25 μM, 6.500 μM and 13.00 μM concentrations of antisense oligonucleotide, as specified in the Table below. After a treatment period of approximately 16 hours, RNA was isolated from the cells and ANGPTL3 mRNA levels were measured by quantitative real-time PCR. Human primer probe set RTS3492_MGB was used to measure mRNA levels. ANGPTL3 mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN. Results are presented as percent inhibition of ANGPTL3, relative to untreated control cells.

The half maximal inhibitory concentration (IC₅₀) of each oligonucleotide is also presented. ANGPTL3 mRNA levels were significantly reduced in a dose-dependent manner in antisense oligonucleotide treated cells.

TABLE 156 IC₅₀ SEQ ID ISIS No 0.813 μM 1.625 μM 3.25 μM 6.50 μM 13.00 μM (μM) NO 233717 0 27 43 66 79 4.4 14 337487 26 49 63 85 94 2.0 28 559277 54 68 70 82 91 <0.8 110 560990 36 61 74 90 96 1.2 111 560992 60 67 76 81 93 <0.8 112 561010 71 77 82 86 94 <0.8 113 561011 80 87 91 95 97 <0.8 114 561022 75 79 84 89 93 <0.8 115 561025 68 82 81 91 96 <0.8 116 561026 72 85 85 89 90 <0.8 117 561208 63 80 87 92 93 <0.8 118 561320 47 60 86 92 96 0.8 119 561343 45 59 79 86 93 0.9 120 561345 38 59 80 88 95 1.1 121 561347 53 63 84 88 97 <0.8 122

TABLE 157 IC₅₀ SEQ ID ISIS No 0.813 μM 1.625 μM 3.25 μM 6.50 μM 13.00 μM (μM) NO 233717 7 19 55 60 77 4.2 14 337487 33 44 69 83 88 2.0 28 560990 36 64 81 87 95 1.1 111 561452 58 69 75 85 88 <0.8 123 561458 69 77 84 91 94 <0.8 124 561460 54 50 72 79 85 <0.8 125 561462 49 72 80 90 92 <0.8 126 561463 63 79 84 92 93 <0.8 127 561478 56 53 80 86 91 <0.8 128 561482 46 69 80 86 91 <0.8 129 561486 56 73 80 91 92 <0.8 130 561487 82 87 88 90 93 <0.8 131 561500 52 60 71 80 91 <0.8 132 561504 49 72 85 91 93 <0.8 133 561621 68 76 85 91 94 <0.8 134

TABLE 158 IC₅₀ SEQ ID ISIS No 0.813 μM 1.625 μM 3.25 μM 6.50 μM 13.00 μM (μM) NO 233717 28 35 48 56 60 4.7 14 337487 43 58 72 82 89 1.0 28 560990 57 73 82 86 96 <0.8 111 561620 51 74 80 85 88 <0.8 135 561622 63 73 85 88 87 <0.8 136 561628 48 69 77 79 80 <0.8 137 561631 60 75 84 86 90 <0.8 138 561644 59 69 77 85 83 <0.8 139 561646 67 81 84 91 92 <0.8 140 561649 70 76 85 89 89 <0.8 141 561650 78 85 88 90 91 <0.8 142 561770 66 81 79 88 91 <0.8 143 561781 65 67 80 81 91 <0.8 144 561791 68 73 83 82 85 <0.8 145 561918 63 71 81 86 92 <0.8 146

TABLE 159 IC₅₀ SEQ ID ISIS No 0.813 μM 1.625 μM 3.25 μM 6.50 μM 13.00 μM (μM) NO 233717 21 26 47 62 69 4.2 14 337487 35 54 73 82 92 1.0 28 560990 42 76 81 88 96 <0.8 111 562078 55 85 86 91 93 <0.8 147 562086 64 83 87 92 93 <0.8 148 562103 72 83 90 90 94 <0.8 149 562110 66 80 83 89 92 <0.8 150 562375 56 61 63 84 90 <0.8 151 562387 67 75 81 90 88 <0.8 152 562396 60 71 80 80 85 <0.8 153 562415 66 73 77 77 81 <0.8 154 562433 68 84 86 90 91 <0.8 155 562436 78 87 87 91 94 <0.8 156 562439 55 66 78 82 93 <0.8 157 562442 55 57 60 76 86 <0.8 158

Example 123: Dose-Dependent Antisense Inhibition of Human ANGPTL3 in Hep3B Cells

Deoxy, MOE, and cEt oligonucleotides from the studies described above exhibiting significant in vitro inhibition of ANGPTL3 mRNA were selected and tested at various doses in Hep3B cells. ISIS 337847, a 5-10-5 MOE gapmer, was also included in the studies. The antisense oligonucleotides were tested in a series of experiments that had similar culture conditions. The results of each experiment are presented in separate tables below.

Cells were plated at a density of 20,000 cells per well and transfected using electroporation with 0.160 μM, 0.481 μM, 1.444 μM, 4.333 μM and 13.00 μM concentrations of antisense oligonucleotide, as specified in the Table below. After a treatment period of approximately 16 hours, RNA was isolated from the cells and ANGPTL3 mRNA levels were measured by quantitative real-time PCR. Human primer probe set RTS3492_MGB was used to measure mRNA levels. ANGPTL3 mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of ANGPTL3, relative to untreated control cells.

The half maximal inhibitory concentration (IC₅₀) of each oligonucleotide is also presented. ANGPTL3 mRNA levels were significantly reduced in a dose-dependent manner in antisense oligonucleotide treated cells.

TABLE 160 IC₅₀ SEQ ID ISIS No 0.160 μM 0.481 μM 1.444 μM 4.333 μM 13.00 μM (μM) NO 337487 0 18 24 49 73 4.1 28 560990 2 27 39 59 80 2.0 111 561076 20 33 59 73 89 1.1 159 561079 24 39 51 72 84 1.0 160 561084 7 17 46 66 87 1.9 161 561085 21 35 55 69 86 1.2 162 561123 20 39 52 72 87 1.1 163 561241 13 22 41 68 86 2.0 164 561256 12 22 35 54 82 2.6 165 561260 22 16 34 54 82 2.6 166 561277 21 21 37 59 69 2.9 167 561288 6 8 23 36 68 6.9 168 561418 25 36 61 79 86 0.9 169 561436 21 40 61 77 88 0.9 170 561443 18 32 52 82 88 1.1 171

TABLE 161 IC₅₀ SEQ ID ISIS No 0.160 μM 0.481 μM 1.444 μM 4.333 μM 13.00 μM (μM) NO 337487 0 8 21 52 81 3.7 28 560990 6 14 40 61 74 3.0 111 561398 3 9 22 64 79 3.0 172 561400 11 28 50 65 83 1.7 173 561528 2 39 59 74 84 1.3 174 561565 18 43 58 75 83 1.0 175 561566 21 29 54 71 79 1.4 176 561567 16 35 56 67 78 1.4 177 561571 18 32 60 80 86 1.1 178 561576 11 12 42 65 77 2.4 179 561689 16 27 52 76 80 1.4 180 561698 1 24 31 61 74 2.9 181 561699 2 19 48 65 81 2.0 182 561722 14 34 59 72 85 1.2 183 561723 7 31 69 71 75 1.4 184

TABLE 162 IC₅₀ SEQ ID ISIS No 0.160 μM 0.481 μM 1.444 μM 4.333 μM 13.00 μM (μM) NO 337487 14 9 9 47 72 5.9 28 560990 13 26 39 58 81 2.0 111 561888 16 19 46 72 84 1.7 185 561897 6 31 50 67 82 2.0 186 561996 19 31 49 59 83 1.6 187 562001 22 46 57 67 89 0.9 188 562024 17 29 59 71 83 1.3 189 562050 21 38 46 62 74 1.6 190 562153 22 35 42 61 71 2.0 191 562155 29 29 50 72 84 1.2 192 562156 15 17 39 60 82 2.3 193 562157 14 15 43 54 75 3.0 194 562181 24 34 58 73 80 1.1 195 562314 22 30 42 54 64 3.1 196 562365 25 27 46 64 77 1.7 197

Example 124: Dose-Dependent Antisense Inhibition of Human ANGPTL3 in Hep3B Cells by MOE Gapmers

MOE gapmers from the Examples above exhibiting significant in vitro inhibition of ANGPTL3 mRNA were selected and tested at various doses in Hep3B cells. Cells were plated at a density of 20,000 cells per well and transfected using electroporation with 0.160 μM, 0.481 μM, 1.444 μM, 4.333 μM and 13.00 μM concentrations of antisense oligonucleotide, as specified in the Table below. After a treatment period of approximately 16 hours, RNA was isolated from the cells and ANGPTL3 mRNA levels were measured by quantitative real-time PCR. Human primer probe set RTS3492_MGB was used to measure mRNA levels. ANGPTL3 mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN. Results are presented as percent inhibition of ANGPTL3, relative to untreated control cells.

The half maximal inhibitory concentration (IC₅₀) of each oligonucleotide is also presented. ANGPTL3 mRNA levels were significantly reduced in a dose-dependent manner in antisense oligonucleotide treated cells.

TABLE 163 IC₅₀ SEQ ID ISIS No Motif 0.16 μM 0.48 μM 1.44 μM 4.33 μM 13.00 μM (μM) NO 233717 5-10-5 0 3 12 38 64 8.0 14 337487 5-10-5 0 0 15 30 66 8.0 28 544120 5-10-5 10 37 62 81 94 1.0 15 567320 5-10-5 0 30 67 84 95 1.1 93 582703 5-10-5 0 18 47 71 83 2.0 198 582705 5-10-5 22 18 46 82 93 1.0 199 582706 5-10-5 2 0 32 67 85 2.6 200 582712 5-10-5 0 0 54 71 89 2.2 201 582713 5-10-5 25 25 52 75 85 1.2 202 582725 5-10-5 0 3 43 62 84 2.7 203 582733 5-10-5 0 30 66 77 87 1.3 204 582743 3-10-4 0 6 37 51 87 2.9 205 582759 3-10-4 0 2 51 76 93 2.0 206 582761 3-10-4 4 38 58 72 87 1.3 207 582774 3-10-4 5 29 46 72 86 1.6 208

Example 125: Dose-Dependent Antisense Inhibition of Human ANGPTL3 in Hep3B Cells by Deoxy, MOE and cEt Oligonucleotides

Deoxy, MOE, and cEt oligonucleotides from the studies described above exhibiting significant in vitro inhibition of ANGPTL3 mRNA were selected and tested at various doses in Hep3B cells. Cells were plated at a density of 20,000 cells per well and transfected using electroporation with 0.111 μM, 0.333 μM, 1.00 μM, 3.00 μM and 9.00 μM concentrations of antisense oligonucleotide, as specified in the Table below. After a treatment period of approximately 16 hours, RNA was isolated from the cells and ANGPTL3 mRNA levels were measured by quantitative real-time PCR. Human primer probe set RTS3492_MGB was used to measure mRNA levels. ANGPTL3 mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN. Results are presented as percent inhibition of ANGPTL3, relative to untreated control cells.

The half maximal inhibitory concentration (IC₅₀) of each oligonucleotide is also presented. ANGPTL3 mRNA levels were significantly reduced in a dose-dependent manner in antisense oligonucleotide treated cells.

TABLE 164 IC₅₀ SEQ ID ISIS No 0.111 μM 0.333 μM 1.00 μM 3.00 μM 9.00 μM (μM) NO 561011 20 39 65 81 94 0.5 114 561026 23 43 65 84 94 0.5 117 561463 26 25 59 76 91 0.7 127 561487 42 61 81 89 95 0.1 131 586661 24 36 46 76 92 0.7 209 586669 31 50 68 85 95 0.3 210 586676 26 50 73 83 95 0.3 211 586688 4 24 51 82 91 0.9 212 586690 19 39 64 84 95 0.5 213 586691 6 37 60 81 93 0.7 214 586701 10 32 55 76 90 0.8 215 586702 16 25 55 69 86 0.9 216 586705 10 30 54 80 89 0.8 217 586707 33 42 71 83 89 0.3 218 586718 38 54 72 78 85 0.2 219

TABLE 165 0.111 0.333 1.00 3.00 9.00 IC₅₀ SEQ ISIS No μM μM μM μM μM (μM) ID NO 561011 13 29 41 76 89 1.0 114 561567 20 46 57 75 78 0.7 177 586692 32 30 71 85 95 0.4 220 586700 3 46 70 82 95 1.0 221 586708 36 46 62 77 86 0.4 222 586744 0 19 54 81 92 1.0 223 586745 35 22 66 78 92 0.5 224 586746 14 30 59 82 92 0.7 225 586755 18 22 53 74 90 0.9 226 586761 26 26 54 73 90 0.8 227 586787 0 38 64 79 90 0.8 228 586796 12 13 56 83 93 0.9 229 586797 4 26 58 82 90 0.9 230 586802 12 28 56 76 81 0.9 231 586804 17 40 65 86 93 0.5 232

TABLE 166 0.111 0.333 1.00 3.00 9.00 IC₅₀ SEQ ISIS No μM μM μM μM μM (μM) ID NO 561011 20 48 75 84 94 0.4 114 561026 31 48 70 88 95 0.3 117 561463 27 40 67 85 94 0.4 127 561487 41 66 84 91 95 0.1 131 586661 36 45 64 82 91 0.3 209 586669 21 55 73 90 96 0.3 210 586676 23 59 77 87 94 0.3 211 586688 25 41 70 82 93 0.4 212 586690 16 45 74 86 92 0.5 213 586691 13 40 65 86 92 0.6 214 586701 22 49 70 82 93 0.4 215 586702 11 31 58 76 92 0.8 216 586705 26 45 66 82 89 0.4 217 586707 28 58 75 85 88 0.3 218 586718 33 59 73 80 88 0.2 219

TABLE 167 0.111 0.333 1.00 3.00 9.00 IC₅₀ SEQ ISIS No μM μM μM μM μM (μM) ID NO 561011 23 41 63 82 92 0.5 114 561567 31 44 65 75 83 0.4 177 586692 16 58 74 89 93 0.4 220 586700 25 62 75 91 94 0.3 221 586708 36 53 72 81 90 0.3 222 586744 30 29 64 75 94 0.6 223 586745 21 44 59 81 89 0.5 224 586746 19 48 57 85 87 0.5 225 586755 6 30 59 78 89 0.8 226 586761 12 29 59 72 87 0.9 227 586787 27 35 64 84 97 0.5 228 586796 31 40 72 91 95 0.3 229 586797 36 47 67 82 88 0.3 230 586802 35 32 61 76 90 0.5 231 586804 35 50 75 91 91 0.2 232

Example 126: Antisense Inhibition of Human ANGPTL3 in huANGPTL3 Transgenic Mice

Antisense oligonucleotides described in the studies above were further evaluated for their ability to reduce human ANGPTL3 mRNA transcript in C57Bl/6 mice with the human ANGPTL3 transgene (Tg mice).

Study 1

Female Tg mice were maintained on a 12-hour light/dark cycle. Animals were acclimated for at least 7 days in the research facility before initiation of the experiment. Antisense oligonucleotides (ASOs) were prepared in buffered saline (PBS) and sterilized by filtering through a 0.2 micron filter. Oligonucleotides were dissolved in 0.9% PBS for injection.

Groups of mice received intraperitoneal injections of 5-10-5 MOE gapmers at a dose of 50 mg/kg once per week for 2 weeks. One group of mice received subcutaneous injections of PBS once weekly for 2 weeks. The PBS-injected group served as the control group to which the corresponding oligonucleotide-treated groups were compared.

RNA Analysis

At the end of the treatment period, RNA was extracted from liver for real-time PCR analysis of measurement of mRNA expression of ANGPTL3 with RTS3492_MGB. mRNA levels were also measured with human primer probe set RTS1984 (forward sequence CTTCAATGAAACGTGGGAGAACT, designated herein as SEQ ID NO: 7; reverse sequence TCTCTAGGCCCAACCAAAATTC, designated herein as SEQ ID NO: 8; probe sequence AAATATGGTTTTGGGAGGCTTGAT, designated herein as SEQ ID NO: 9). Results are presented as percent change of mRNA, relative to PBS control, normalized with RIBOGREEN®. As shown in the Table below, treatment with ISIS antisense oligonucleotides resulted in significant reduction of ANGPTL3 mRNA in comparison to the PBS control.

TABLE 168 Percent inhibition of ANGPTL3 mRNA in transgenic mouse liver relative to the PBS control SEQ ISIS No RTS3492_MGB RTS1984 ID NO 233710 91 94 233 233717 49 58 14 337477 76 82 234 337478 52 65 235 337479 53 76 236 337487 80 92 28 Protein Analysis

Human ANGPTL3 protein levels were quantified using a commercially available ELISA kit (Catalog #DANL30 by R&D Systems, Minneapolis, Minn.) with transgenic plasma samples diluted 1:20,000 using the manufacturer described protocol. The results are presented in the Table below. The results indicate that treatment with ISIS oligonucleotides resulted in reduced ANGPTL3 protein levels.

TABLE 169 Percent inhibition of plasma protein levels in the transgenic mouse SEQ ISIS No % ID NO 233710 92 233 233717 47 14 337477 68 234 337478 36 235 337479 48 236 337487 78 28 Plasma Chemistry Markers

To evaluate the effect of ISIS oligonucleotides on day 10, plasma levels of transaminases (ALT and AST) were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.). The results are presented in the Table below. ISIS oligonucleotides that caused changes in the levels of any of these liver function markers outside the expected range for antisense oligonucleotides were excluded in further studies.

TABLE 170 Plasma transaminase levels (IU/L) in transgenic mice on day 10 SEQ ALT AST ID NO PBS 27 36 ISIS 233710 19 37 233 ISIS 233717 16 32 14 ISIS 337477 22 35 234 ISIS 337478 23 49 235 ISIS 337479 21 29 236 ISIS 337487 19 35 28 Study 2

Male Tg mice were maintained on a 12-hour light/dark cycle. Animals were acclimated for at least 7 days in the research facility before initiation of the experiment. Antisense oligonucleotides (ASOs) were prepared in buffered saline (PBS) and sterilized by filtering through a 0.2 micron filter. Oligonucleotides were dissolved in 0.9% PBS for injection.

Groups of mice received intraperitoneal injections of 5-10-5 MOE gapmers at a dose of 50 mg/kg once per week for 2 weeks. One group of mice received subcutaneous injections of PBS once weekly for 2 weeks. The PBS-injected groups served as the control groups to which the corresponding oligonucleotide-treated groups were compared.

RNA Analysis

At the end of the treatment period, RNA was extracted from liver for real-time PCR analysis of measurement of mRNA expression of ANGPTL3 with RTS1984. Results are presented as percent change of mRNA, relative to PBS control, normalized with RIBOGREEN. As shown in the Table below, treatment with ISIS antisense oligonucleotides resulted in significant reduction of ANGPTL3 mRNA in comparison to the PBS control.

TABLE 171 Percent inhibition of ANGPTL3 mRNA in transgenic mouse liver relative to the PBS control SEQ ISIS No % ID NO 233710 81 233 337487 92 28 544145 98 16 544162 75 18 544199 97 20 560306 90 34 560400 97 35 560401 95 36 560402 98 37 560469 98 38 560735 87 49 567320 95 93 567321 93 94 Protein Analysis

Human ANGPTL3 protein levels were quantified using a commercially available ELISA kit (Catalog #DANL30 by R&D Systems, Minneapolis, Minn.) with transgenic plasma samples diluted 1:20,000 using the manufacturer described protocol. The results are presented in the Table below. The results indicate that treatment with ISIS oligonucleotides resulted in reduced ANGPTL3 protein levels.

TABLE 172 Percent inhibition of plasma protein levels in the transgenic mouse SEQ ISIS No % ID NO 233710 96 233 337487 78 28 544145 96 16 544162 97 18 544199 98 20 560306 97 34 560400 98 35 560401 97 36 560402 94 37 560469 96 38 560735 91 49 567320 98 93 567321 96 94 Plasma Chemistry Markers

To evaluate the effect of ISIS oligonucleotides on day 8, plasma levels of transaminases (ALT and AST) were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.). The results are presented in the Table below. ISIS oligonucleotides that caused changes in the levels of any of these liver function markers outside the expected range for antisense oligonucleotides were excluded in further studies.

TABLE 173 Plasma transaminase levels (IU/L) in transgenic mice on day 8 SEQ ALT AST ID NO PBS 29 44 ISIS 233710 29 47 233 ISIS 337487 22 36 28 ISIS 544145 29 45 16 ISIS 544162 31 62 18 ISIS 544199 29 51 20 ISIS 560306 23 42 34 ISIS 560400 24 52 35 ISIS 560401 20 38 36 ISIS 560402 29 49 37 ISIS 560469 22 50 38 ISIS 560735 20 38 49 ISIS 567320 49 71 93 ISIS 567321 20 44 94 Study 3

Male and female Tg mice were maintained on a 12-hour light/dark cycle. Animals were acclimated for at least 7 days in the research facility before initiation of the experiment. Antisense oligonucleotides (ASOs) were prepared in buffered saline (PBS) and sterilized by filtering through a 0.2 micron filter. Oligonucleotides were dissolved in 0.9% PBS for injection.

Groups of mice received intraperitoneal injections of 5-10-5 MOE gapmers at a dose of 2.5 mg/kg, 12.5 mg/kg, or 25 mg/kg once per week for 3 weeks. One group of mice received subcutaneous injections of PBS once weekly for 2 weeks. The PBS-injected groups served as the control groups to which the corresponding oligonucleotide-treated groups were compared.

RNA Analysis

At the end of the treatment period, RNA was extracted from liver for real-time PCR analysis of measurement of mRNA expression of ANGPTL3 with hANGPTL3_LTS01022 (forward sequence AAATTTTAGCCAATGGCCTCC, designated herein as SEQ ID NO: 10; reverse sequence TGTCATTAATTTGGCCCTTCG, designated herein as SEQ ID NO: 11; probe sequence TCAGTTGGGACATGGTCTTAAAGACTTTGTCC, designated herein as SEQ ID NO: 12). Results are presented as percent change of mRNA, relative to PBS control, normalized with RIBOGREEN. As shown in the Table below, treatment with ISIS antisense oligonucleotides resulted in significant reduction of ANGPTL3 mRNA in comparison to the PBS control. The ED₅₀ of each gapmer is also presented in the Table below. ‘n.d.’ indicates that the ED₅₀ could not be determined.

TABLE 174 Percent inhibition of ANGPTL3 mRNA in transgenic mouse liver relative to the PBS control Dose SEQ ISIS No (mg/kg) % ED₅₀ ID NO 233710 25 88 8 233 12.5 79 2.5 0 544145 25 90 4 16 12.5 74 2.5 39 544162 25 53 9 18 12.5 63 2.5 39 544199 25 81 7 20 12.5 82 2.5 7 560306 25 0 n.d. 34 12.5 0 2.5 0 560400 25 87 5 35 12.5 76 2.5 24 560401 25 89 8 36 12.5 62 2.5 5 560469 25 73 3 38 12.5 78 2.5 50 560735 25 26 31 49 12.5 37 2.5 51 567320 25 74 12 93 12.5 37 2.5 32 567321 25 75 11 94 12.5 61 2.5 0 Protein Analysis

Human ANGPTL3 protein levels were quantified using a commercially available ELISA kit (Catalog #DANL30 by R&D Systems, Minneapolis, Minn.) with transgenic plasma samples diluted 1:20,000 using the manufacturer described protocol. The results are presented in the Table below. The results indicate that treatment with ISIS oligonucleotides resulted in reduced ANGPTL3 protein levels. ‘n.d.’ indicates that the ED₅₀ could not be determined.

TABLE 175 Percent inhibition of plasma protein levels in the transgenic mouse Dose SEQ ISIS No (mg/kg) % ED₅₀ ID NO 233710 25 80 11 233 12.5 56 2.5 0 544145 25 88 9 16 12.5 64 2.5 0 544162 25 56 15 18 12.5 46 2.5 24 544199 25 73 6 20 12.5 73 2.5 31 560306 25 63 n.d. 34 12.5 55 2.5 53 560400 25 88 6 35 12.5 73 2.5 20 560401 25 88 10 36 12.5 61 2.5 0 560469 25 75 4 38 12.5 70 2.5 52 560735 25 27 34 49 12.5 37 2.5 34 567320 25 69 10 93 12.5 44 2.5 39 567321 25 68 12 94 12.5 62 2.5 1 Plasma Chemistry Markers

To evaluate the effect of ISIS oligonucleotides on day 17, plasma levels of transaminases (ALT and AST) were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.). The results are presented in the Table below. ISIS oligonucleotides that caused changes in the levels of any of these liver function markers outside the expected range for antisense oligonucleotides were excluded in further studies.

TABLE 176 Plasma transaminase levels (IU/L) in transgenic mice on day 17 Dose SEQ (mg/kg) ALT AST ID NO PBS — 25 38 ISIS 25 27 40 233 233710 12.5 24 45 2.5 23 36 ISIS 25 30 56 16 544145 12.5 25 52 2.5 28 43 ISIS 25 28 52 18 544162 12.5 36 53 2.5 28 50 ISIS 25 24 47 20 544199 12.5 23 60 2.5 24 44 ISIS 25 21 45 34 560306 12.5 24 49 2.5 24 47 ISIS 25 22 38 35 560400 12.5 21 53 2.5 23 52 ISIS 25 36 80 36 560401 12.5 27 75 2.5 22 49 ISIS 25 24 121 38 560469 12.5 23 53 2.5 21 88 ISIS 25 20 48 49 560735 12.5 22 138 2.5 24 78 ISIS 25 21 65 93 567320 12.5 20 58 2.5 23 46 ISIS 25 23 62 94 567321 12.5 21 49 2.5 24 67 Study 4

Male and female Tg mice were maintained on a 12-hour light/dark cycle. Animals were acclimated for at least 7 days in the research facility before initiation of the experiment. Antisense oligonucleotides (ASOs) were prepared in buffered saline (PBS) and sterilized by filtering through a 0.2 micron filter. Oligonucleotides were dissolved in 0.9% PBS for injection.

Groups of mice received intraperitoneal injections of 5-10-5 MOE gapmers at a dose of 25 mg/kg once per week for 2 weeks. One group of mice received subcutaneous injections of PBS once weekly for 2 weeks. The PBS-injected group served as the control group to which the corresponding oligonucleotide-treated groups were compared.

RNA Analysis

At the end of the treatment period, RNA was extracted from liver for real-time PCR analysis of measurement of mRNA expression of ANGPTL3 with hANGPTL3_LTS01022. Results are presented as percent change of mRNA, relative to PBS control, normalized with RIBOGREEN. As shown in the Table below, treatment with ISIS antisense oligonucleotides resulted in significant reduction of ANGPTL3 mRNA in comparison to the PBS control.

TABLE 177 Percent inhibition of ANGPTL3 mRNA in transgenic mouse liver relative to the PBS control SEQ ISIS No % ID NO 233710 68 233 544120 63 15 544199 82 20 544355 0 21 560268 36 32 560470 47 39 560471 67 40 560474 57 41 560566 45 42 560567 68 43 560607 37 46 560608 15 47 560744 25 51 560778 32 52 560811 27 54 560925 0 56 563639 5 79 567291 8 91 567330 30 95 568049 48 101 568146 26 104 Plasma Chemistry Markers

To evaluate the effect of ISIS oligonucleotides on day 10, plasma levels of transaminases (ALT and AST) were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.). The results are presented in the Table below. ISIS oligonucleotides that caused changes in the levels of any of these liver function markers outside the expected range for antisense oligonucleotides were excluded in further studies.

TABLE 178 Plasma transaminase levels (IU/L) in transgenic mice on day 10 SEQ ALT AST ID NO PBS 29 41 ISIS 233710 29 48 233 ISIS 544120 24 35 15 ISIS 544199 27 57 20 ISIS 544355 23 44 21 ISIS 560268 23 42 32 ISIS 560470 26 42 39 ISIS 560471 21 50 40 ISIS 560474 20 33 41 ISIS 560566 27 102 42 ISIS 560567 20 37 43 ISIS 560607 25 47 46 ISIS 560608 20 49 47 ISIS 560744 26 66 51 ISIS 560778 24 87 52 ISIS 560811 21 63 54 ISIS 560925 25 115 56 ISIS 563639 20 43 79 ISIS 567291 20 67 91 ISIS 567330 29 78 95 ISIS 568049 25 63 101 ISIS 568146 28 140 104 Study 5

Male and female Tg mice were maintained on a 12-hour light/dark cycle. Animals were acclimated for at least 7 days in the research facility before initiation of the experiment. Antisense oligonucleotides (ASOs) were prepared in buffered saline (PBS) and sterilized by filtering through a 0.2 micron filter. Oligonucleotides were dissolved in 0.9% PBS for injection.

Groups of mice received intraperitoneal injections of 5-10-5 MOE gapmers or deoxy, MOE, and cEt gapmers at a dose of 25 mg/kg once per week for 2 weeks. One group of mice received subcutaneous injections of PBS once weekly for 2 weeks. The PBS-injected group served as the control group to which the corresponding oligonucleotide-treated groups were compared.

RNA Analysis

At the end of the treatment period, RNA was extracted from liver for real-time PCR analysis of measurement of mRNA expression of ANGPTL3 with RTS1984. Results are presented as percent change of mRNA, relative to PBS control, normalized with RIBOGREEN®. As shown in the Table below, treatment with ISIS antisense oligonucleotides resulted in significant reduction of ANGPTL3 mRNA in comparison to the PBS control.

TABLE 179 Percent inhibition of ANGPTL3 mRNA in transgenic mouse liver relative to the PBS control SEQ ISIS No Chemistry % ID NO 233710 5-10-5 MOE 79 233 544156 5-10-5 MOE 92 17 559277 Deoxy, MOE and cEt 75 110 560265 5-10-5 MOE 52 31 560285 5-10-5 MOE 42 33 560574 5-10-5 MOE 93 44 560847 5-10-5 MOE 61 69 560992 Deoxy, MOE and cEt 80 112 561010 Deoxy, MOE and cEt 66 113 561011 Deoxy, MOE and cEt 96 114 561022 Deoxy, MOE and cEt 79 115 561025 Deoxy, MOE and cEt 57 116 563580 5-10-5 MOE 80 77 567115 5-10-5 MOE 78 88 567233 5-10-5 MOE 91 90 Plasma Chemistry Markers

To evaluate the effect of ISIS oligonucleotides on day 9, plasma levels of transaminases (ALT and AST) were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.). The results are presented in the Table below. ISIS oligonucleotides that caused changes in the levels of any of these liver function markers outside the expected range for antisense oligonucleotides were excluded in further studies.

TABLE 180 Plasma transaminase levels (IU/L) in transgenic mice on day 9 SEQ Chemistry ALT AST ID NO PBS — 48 65 ISIS 233710 5-10-5 MOE 24 43 233 ISIS 544156 5-10-5 MOE 29 44 17 ISIS 559277 Deoxy, MOE and cEt 22 38 110 ISIS 560265 5-10-5 MOE 28 83 31 ISIS 560285 5-10-5 MOE 29 44 33 ISIS 560574 5-10-5 MOE 24 54 44 ISIS 560847 5-10-5 MOE 25 45 69 ISIS 560992 Deoxy, MOE and cEt 32 128 112 ISIS 561010 Deoxy, MOE and cEt 22 51 113 ISIS 561011 Deoxy, MOE and cEt 28 43 114 ISIS 561022 Deoxy, MOE and cEt 51 85 115 ISIS 561025 Deoxy, MOE and cEt 22 48 116 ISIS 563580 5-10-5 MOE 28 109 77 ISIS 567115 5-10-5 MOE 21 42 88 ISIS 567233 5-10-5 MOE 22 73 90 Study 6

Male and female Tg mice were maintained on a 12-hour light/dark cycle. Animals were acclimated for at least 7 days in the research facility before initiation of the experiment. Antisense oligonucleotides (ASOs) were prepared in buffered saline (PBS) and sterilized by filtering through a 0.2 micron filter. Oligonucleotides were dissolved in 0.9% PBS for injection.

Groups of mice received intraperitoneal injections of deoxy, MOE, and cEt oligonucleotides at a dose of 25 mg/kg once per week for 2 weeks. ISIS 233710, a 5-10-5 MOE gapmer, was also included as a benchmark. One group of mice received subcutaneous injections of PBS once weekly for 2 weeks. The PBS-injected group served as the control group to which the corresponding oligonucleotide-treated groups were compared.

RNA Analysis

At the end of the treatment period, RNA was extracted from liver for real-time PCR analysis of measurement of mRNA expression of ANGPTL3 with hANGPTL3_LTS01022. Results are presented as percent change of mRNA, relative to PBS control, normalized with RIBOGREEN. As shown in the Table below, treatment with several of the ISIS antisense oligonucleotides resulted in significant reduction of ANGPTL3 mRNA in comparison to the PBS control.

TABLE 181 Percent inhibition of ANGPTL3 mRNA in transgenic mouse liver relative to the PBS control SEQ ISIS No Chemistry % ID NO 233710 5-10-5 MOE 68 233 561026 Deoxy, MOE and cEt 94 117 561079 Deoxy, MOE and cEt 51 160 561084 Deoxy, MOE and cEt 56 161 561123 Deoxy, MOE and cEt 47 163 561208 Deoxy, MOE and cEt 42 118 561241 Deoxy, MOE and cEt 13 164 561400 Deoxy, MOE and cEt 31 173 561418 Deoxy, MOE and cEt 32 169 561436 Deoxy, MOE and cEt 67 170 561443 Deoxy, MOE and cEt 12 171 561458 Deoxy, MOE and cEt 57 124 Protein Analysis

Human ANGPTL3 protein levels were quantified using a commercially available ELISA kit (Catalog #DANL30 by R&D Systems, Minneapolis, Minn.) with transgenic plasma samples diluted 1:20,000 using the manufacturer described protocol. The results are presented in the Table below. The results indicate that treatment with several of the ISIS oligonucleotides resulted in reduced ANGPTL3 protein levels.

TABLE 182 Percent inhibition of plasma protein levels in the transgenic mouse SEQ ID ISIS No Chemistry % NO 233710 5-10-5 MOE 82 233 561026 Deoxy, MOE and cEt 92 117 561079 Deoxy, MOE and cEt 80 160 561084 Deoxy, MOE and cEt 89 161 561123 Deoxy, MOE and cEt 62 163 561208 Deoxy, MOE and cEt 0 118 561241 Deoxy, MOE and cEt 36 164 561400 Deoxy, MOE and cEt 60 173 561418 Deoxy, MOE and cEt 42 169 561436 Deoxy, MOE and cEt 46 170 561443 Deoxy, MOE and cEt 27 171 561458 Deoxy, MOE and cEt 71 124 Plasma Chemistry Markers

To evaluate the effect of ISIS oligonucleotides on day 10, plasma levels of transaminases (ALT and AST) were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.). The results are presented in the Table below. ISIS oligonucleotides that caused changes in the levels of any of these liver function markers outside the expected range for antisense oligonucleotides were excluded in further studies.

TABLE 183 Plasma transaminase levels (IU/L) in transgenic mice on day 10 SEQ ID Chemistry ALT AST NO PBS — 41 64 ISIS 233710 5-10-5 MOE 25 74 233 ISIS 561026 Deoxy, MOE and cEt 30 67 117 ISIS 561079 Deoxy, MOE and cEt 42 62 160 ISIS 561084 Deoxy, MOE and cEt 70 101 161 ISIS 561123 Deoxy, MOE and cEt 24 41 163 ISIS 561208 Deoxy, MOE and cEt 203 168 118 ISIS 561241 Deoxy, MOE and cEt 26 47 164 ISIS 561400 Deoxy, MOE and cEt 27 83 173 ISIS 561418 Deoxy, MOE and cEt 58 164 169 ISIS 561436 Deoxy, MOE and cEt 24 42 170 ISIS 561443 Deoxy, MOE and cEt 27 91 171 ISIS 561458 Deoxy, MOE and cEt 30 144 124 Study 7

Male and female Tg mice were maintained on a 12-hour light/dark cycle. Animals were acclimated for at least 7 days in the research facility before initiation of the experiment. Antisense oligonucleotides (ASOs) were prepared in buffered saline (PBS) and sterilized by filtering through a 0.2 micron filter. Oligonucleotides were dissolved in 0.9% PBS for injection.

Groups of mice received intraperitoneal injections of deoxy, MOE, and cEt oligonucleotides at a dose of 25 mg/kg once per week for 2 weeks. ISIS 233710, a 5-10-5 MOE gapmer, was also included as a benchmark. One group of mice received subcutaneous injections of PBS once weekly for 2 weeks. The PBS-injected group served as the control group to which the corresponding oligonucleotide-treated groups were compared.

RNA Analysis

At the end of the treatment period, RNA was extracted from liver for real-time PCR analysis of measurement of mRNA expression of ANGPTL3 with hANGPTL3_LTS01022. Results are presented as percent change of mRNA, relative to PBS control, normalized with RIBOGREEN®. As shown in the Table below, treatment with ISIS antisense oligonucleotides resulted in significant reduction of ANGPTL3 mRNA in comparison to the PBS control.

TABLE 184 Percent inhibition of ANGPTL3 mRNA in transgenic mouse liver relative to the PBS control SEQ ID ISIS No Chemistry % NO 233710 5-10-5 MOE 80 233 561462 Deoxy, MOE and cEt 84 126 561463 Deoxy, MOE and cEt 84 127 561486 Deoxy, MOE and cEt 74 130 561487 Deoxy, MOE and cEt 82 131 561504 Deoxy, MOE and cEt 51 133 561528 Deoxy, MOE and cEt 87 174 561565 Deoxy, MOE and cEt 94 175 561566 Deoxy, MOE and cEt 76 176 561571 Deoxy, MOE and cEt 51 178 561621 Deoxy, MOE and cEt 93 134 561646 Deoxy, MOE and cEt 39 140 561649 Deoxy, MOE and cEt 93 141 561650 Deoxy, MOE and cEt 82 142 561689 Deoxy, MOE and cEt 51 180 561722 Deoxy, MOE and cEt 88 183 561723 Deoxy, MOE and cEt 85 184 561770 Deoxy, MOE and cEt 70 143 562024 Deoxy, MOE and cEt 82 189 Protein Analysis

Human ANGPTL3 protein levels were quantified using a commercially available ELISA kit (Catalog #DANL30 by R&D Systems, Minneapolis, Minn.) with transgenic plasma samples diluted 1:20,000 using the manufacturer described protocol. The results are presented in the Table below. The results indicate that treatment with some of the ISIS oligonucleotides resulted in reduced ANGPTL3 levels. In this case, ‘0’ value implies that treatment with the ISIS oligonucleotide did not inhibit expression; in some instances, increased levels of expression may have been recorded.

TABLE 185 Percent inhibition of plasma protein levels in the transgenic mouse SEQ ID ISIS No Chemistry % NO 233710 5-10-5 MOE 60 233 561462 Deoxy, MOE and cEt 62 126 561463 Deoxy, MOE and cEt 59 127 561486 Deoxy, MOE and cEt 0 130 561487 Deoxy, MOE and cEt 0 131 561504 Deoxy, MOE and cEt 0 133 561528 Deoxy, MOE and cEt 0 174 561565 Deoxy, MOE and cEt 71 175 561566 Deoxy, MOE and cEt 0 176 561571 Deoxy, MOE and cEt 0 178 561621 Deoxy, MOE and cEt 72 134 561646 Deoxy, MOE and cEt 0 140 561649 Deoxy, MOE and cEt 63 141 561650 Deoxy, MOE and cEt 0 142 561689 Deoxy, MOE and cEt 0 180 561722 Deoxy, MOE and cEt 0 183 561723 Deoxy, MOE and cEt 0 184 561770 Deoxy, MOE and cEt 0 143 562024 Deoxy, MOE and cEt 0 189 Plasma Chemistry Markers

To evaluate the effect of ISIS oligonucleotides on day 9, plasma levels of transaminases (ALT and AST) were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.). The results are presented in the Table below. ISIS oligonucleotides that caused changes in the levels of any of these liver function markers outside the expected range for antisense oligonucleotides were excluded in further studies.

TABLE 186 Plasma transaminase levels (IU/L) in transgenic mice on day 9 SEQ ID Chemistry ALT AST NO PBS — 35 72 ISIS 233710 5-10-5 MOE 23 39 233 ISIS 561462 Deoxy, MOE and cEt 26 56 126 ISIS 561463 Deoxy, MOE and cEt 34 61 127 ISIS 561486 Deoxy, MOE and cEt 23 61 130 ISIS 561487 Deoxy, MOE and cEt 21 64 131 ISIS 561504 Deoxy, MOE and cEt 26 66 133 ISIS 561528 Deoxy, MOE and cEt 26 86 174 ISIS 561565 Deoxy, MOE and cEt 24 43 175 ISIS 561566 Deoxy, MOE and cEt 23 62 176 ISIS 561571 Deoxy, MOE and cEt 26 68 178 ISIS 561621 Deoxy, MOE and cEt 26 96 134 ISIS 561646 Deoxy, MOE and cEt 24 77 140 ISIS 561649 Deoxy, MOE and cEt 22 94 141 ISIS 561650 Deoxy, MOE and cEt 34 121 142 ISIS 561689 Deoxy, MOE and cEt 24 73 180 ISIS 561722 Deoxy, MOE and cEt 34 89 183 ISIS 561723 Deoxy, MOE and cEt 24 65 184 ISIS 561770 Deoxy, MOE and cEt 22 69 143 ISIS 562024 Deoxy, MOE and cEt 32 162 189 Study 8

Male and female Tg mice were maintained on a 12-hour light/dark cycle. Animals were acclimated for at least 7 days in the research facility before initiation of the experiment. Antisense oligonucleotides (ASOs) were prepared in buffered saline (PBS) and sterilized by filtering through a 0.2 micron filter. Oligonucleotides were dissolved in 0.9% PBS for injection.

Groups of mice received intraperitoneal injections of deoxy, MOE, and cEt oligonucleotides at a dose of 25 mg/kg once per week for 2 weeks. ISIS 233710, a 5-10-5 MOE gapmer, was also included as a benchmark. One group of mice received subcutaneous injections of PBS once weekly for 2 weeks. The PBS-injected group served as the control group to which the corresponding oligonucleotide-treated groups were compared.

RNA Analysis

At the end of the treatment period, RNA was extracted from liver for real-time PCR analysis of measurement of mRNA expression of ANGPTL3 with hANGPTL3_LTS01022. Results are presented as percent change of mRNA, relative to PBS control, normalized with RIBOGREEN. As shown in the Table below, treatment with ISIS antisense oligonucleotides resulted in significant reduction of ANGPTL3 mRNA in comparison to the PBS control.

TABLE 187 Percent inhibition of ANGPTL3 mRNA in transgenic mouse liver relative to the PBS control SEQ ID ISIS No Chemistry % NO 233710 5-10-5 MOE 99 233 562078 Deoxy, MOE and cEt 73 147 562086 Deoxy, MOE and cEt 85 148 562103 Deoxy, MOE and cEt 58 149 562110 Deoxy, MOE and cEt 94 150 562155 Deoxy, MOE and cEt 85 192 562181 Deoxy, MOE and cEt 79 195 562433 Deoxy, MOE and cEt 59 155 562436 Deoxy, MOE and cEt 99 156 586669 Deoxy, MOE and cEt 95 210 586676 Deoxy, MOE and cEt 80 211 Protein Analysis

Human ANGPTL3 protein levels were quantified using a commercially available ELISA kit (Catalog #DANL30 by R&D Systems, Minneapolis, Minn.) with transgenic plasma samples diluted 1:20,000 using the manufacturer described protocol. The results are presented in the Table below. The results indicate that treatment with the ISIS oligonucleotides resulted in reduced ANGPTL3 levels.

TABLE 188 Percent inhibition of plasma protein levels in the transgenic mouse SEQ ID ISIS No Chemistry % NO 233710 5-10-5 MOE 69 233 562078 Deoxy, MOE and cEt 44 147 562086 Deoxy, MOE and cEt 91 148 562103 Deoxy, MOE and cEt 26 149 562110 Deoxy, MOE and cEt 68 150 562155 Deoxy, MOE and cEt 75 192 562181 Deoxy, MOE and cEt 86 195 562433 Deoxy, MOE and cEt 80 155 562436 Deoxy, MOE and cEt 98 156 586669 Deoxy, MOE and cEt 98 210 586676 Deoxy, MOE and cEt 95 211 Plasma Chemistry Markers

To evaluate the effect of ISIS oligonucleotides on day 8, plasma levels of transaminases (ALT and AST) were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.). The results are presented in the Table below. ISIS oligonucleotides that caused changes in the levels of any of these liver function markers outside the expected range for antisense oligonucleotides were excluded in further studies.

TABLE 189 Plasma transaminase levels (IU/L) in transgenic mice on day 8 SEQ ID Chemistry ALT AST NO PBS — 44 248 ISIS 233710 5-10-5 MOE 27 52 233 ISIS 562078 Deoxy, MOE and cEt 41 130 147 ISIS 562086 Deoxy, MOE and cEt 30 62 148 ISIS 562103 Deoxy, MOE and cEt 35 99 149 ISIS 562110 Deoxy, MOE and cEt 30 161 150 ISIS 562155 Deoxy, MOE and cEt 68 622 192 ISIS 562181 Deoxy, MOE and cEt 37 168 195 ISIS 562433 Deoxy, MOE and cEt 33 209 155 ISIS 562436 Deoxy, MOE and cEt 30 93 156 ISIS 586669 Deoxy, MOE and cEt 27 141 210 ISIS 586676 Deoxy, MOE and cEt 22 60 211 Study 9

Male and female Tg mice were maintained on a 12-hour light/dark cycle. Animals were acclimated for at least 7 days in the research facility before initiation of the experiment. Antisense oligonucleotides (ASOs) were prepared in buffered saline (PBS) and sterilized by filtering through a 0.2 micron filter. Oligonucleotides were dissolved in 0.9% PBS for injection.

Groups of mice received intraperitoneal injections of deoxy, MOE, and cEt oligonucleotides at a dose of 25 mg/kg once per week for 2 weeks. ISIS 233710, a 5-10-5 MOE gapmer, was also included as a benchmark. One group of mice received subcutaneous injections of PBS once weekly for 2 weeks. The PBS-injected group served as the control group to which the corresponding oligonucleotide-treated groups were compared.

RNA Analysis

At the end of the treatment period, RNA was extracted from liver for real-time PCR analysis of measurement of mRNA expression of ANGPTL3 with hANGPTL3_LTS01022. Results are presented as percent change of mRNA, relative to PBS control, normalized with RIBOGREEN. As shown in the Table below, treatment with some of the ISIS antisense oligonucleotides resulted in significant reduction of ANGPTL3 mRNA in comparison to the PBS control. In this case, ‘0’ value implies that treatment with the ISIS oligonucleotide did not inhibit expression; in some instances, increased levels of expression may have been recorded.

TABLE 190 Percent inhibition of ANGPTL3 mRNA in transgenic mouse liver relative to the PBS control SEQ ID ISIS No Chemistry % NO 233710 5-10-5 MOE 84 233 586690 Deoxy, MOE and cEt 45 213 586692 Deoxy, MOE and cEt 45 220 586700 Deoxy, MOE and cEt 46 221 586707 Deoxy, MOE and cEt 88 218 586708 Deoxy, MOE and cEt 73 222 586718 Deoxy, MOE and cEt 20 219 586744 Deoxy, MOE and cEt 0 223 586745 Deoxy, MOE and cEt 0 224 586755 Deoxy, MOE and cEt 75 226 586761 Deoxy, MOE and cEt 66 227 586787 Deoxy, MOE and cEt 47 228 586796 Deoxy, MOE and cEt 88 229 586797 Deoxy, MOE and cEt 81 230 586802 Deoxy, MOE and cEt 33 231 586804 Deoxy, MOE and cEt 60 232 Protein Analysis

Human ANGPTL3 protein levels were quantified using a commercially available ELISA kit (Catalog #DANL30 by R&D Systems, Minneapolis, Minn.) with transgenic plasma samples diluted 1:20,000 using the manufacturer described protocol. The results are presented in the Table below. The results indicate that treatment with some of the ISIS oligonucleotides resulted in reduced ANGPTL3 levels. In this case, ‘0’ value implies that treatment with the ISIS oligonucleotide did not inhibit expression; in some instances, increased levels of expression may have been recorded.

TABLE 191 Percent inhibition of plasma protein levels in the transgenic mouse SEQ ID ISIS No Chemistry % NO 233710 5-10-5 MOE 80 233 586690 Deoxy, MOE and cEt 21 213 586692 Deoxy, MOE and cEt 46 220 586700 Deoxy, MOE and cEt 0 221 586707 Deoxy, MOE and cEt 84 218 586708 Deoxy, MOE and cEt 32 222 586718 Deoxy, MOE and cEt 0 219 586744 Deoxy, MOE and cEt 0 223 586745 Deoxy, MOE and cEt 0 224 586755 Deoxy, MOE and cEt 0 226 586761 Deoxy, MOE and cEt 0 227 586787 Deoxy, MOE and cEt 0 228 586796 Deoxy, MOE and cEt 40 229 586797 Deoxy, MOE and cEt 50 230 586802 Deoxy, MOE and cEt 0 231 586804 Deoxy, MOE and cEt 0 232 Plasma Chemistry Markers

To evaluate the effect of ISIS oligonucleotides on day 9, plasma levels of transaminases (ALT and AST) were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.). The results are presented in the Table below. ISIS oligonucleotides that caused changes in the levels of any of these liver function markers outside the expected range for antisense oligonucleotides were excluded in further studies.

TABLE 192 Plasma transaminase levels (IU/L) in transgenic mice on day 9 SEQ ID Chemistry ALT AST NO PBS — 28 73 ISIS 233710 5-10-5 MOE 22 86 233 ISIS 586690 Deoxy, MOE and cEt 42 120 213 ISIS 586692 Deoxy, MOE and cEt 22 45 220 ISIS 586700 Deoxy, MOE and cEt 24 84 221 ISIS 586707 Deoxy, MOE and cEt 26 44 218 ISIS 586708 Deoxy, MOE and cEt 22 48 222 ISIS 586718 Deoxy, MOE and cEt 22 39 219 ISIS 586744 Deoxy, MOE and cEt 26 83 223 ISIS 586745 Deoxy, MOE and cEt 25 56 224 ISIS 586746 Deoxy, MOE and cEt 77 77 225 ISIS 586755 Deoxy, MOE and cEt 28 148 226 ISIS 586761 Deoxy, MOE and cEt 36 126 227 ISIS 586787 Deoxy, MOE and cEt 23 88 228 ISIS 586796 Deoxy, MOE and cEt 32 148 229 ISIS 586797 Deoxy, MOE and cEt 29 151 230 ISIS 586802 Deoxy, MOE and cEt 35 200 231 ISIS 586804 Deoxy, MOE and cEt 24 87 232 Study 10

Male and female Tg mice were maintained on a 12-hour light/dark cycle. Animals were acclimated for at least 7 days in the research facility before initiation of the experiment. Antisense oligonucleotides (ASOs) were prepared in buffered saline (PBS) and sterilized by filtering through a 0.2 micron filter. Oligonucleotides were dissolved in 0.9% PBS for injection.

Groups of mice received intraperitoneal injections of 5-10-5 MOE gapmers or deoxy, MOE and cEt oligonucleotides at a dose of 5 mg/kg, 12.5 mg/kg, or 25 mg/kg once per week for 2 weeks. One group of mice received subcutaneous injections of PBS once weekly for 2 weeks. The PBS-injected group served as the control group to which the corresponding oligonucleotide-treated groups were compared.

RNA Analysis

At the end of the treatment period, RNA was extracted from liver for real-time PCR analysis of measurement of mRNA expression of ANGPTL3 with hANGPTL3_LTS01022, and also with RTS3492_MGB. Results are presented as percent change of mRNA, relative to PBS control, normalized with RIBOGREEN®. As shown in the Table below, treatment with some of the ISIS antisense oligonucleotides resulted in reduction of ANGPTL3 mRNA in comparison to the PBS control.

TABLE 193 Percent inhibition of ANGPTL3 mRNA in transgenic mouse liver relative to the PBS control Dose SEQ ID ISIS No Chemistry (mg/kg) RTS3492_MGB hANGPTL3_LTS01022 NO 233710 5-10-5 MOE 25 0 8 233 12.5 24 22 5 12 22 544199 5-10-5 MOE 25 63 59 20 12.5 43 43 5 17 24 559277 Deoxy, MOE 25 37 46 110 and cEt 12.5 0 0 5 0 0 560400 5-10-5 MOE 25 45 48 35 12.5 36 50 5 0 0 561010 Deoxy, MOE 25 5 37 113 and cEt 12.5 0 6 5 0 0 563580 5-10-5 MOE 25 56 59 77 12.5 43 44 5 5 9 567320 5-10-5 MOE 25 47 50 93 12.5 0 0 5 0 0 567321 5-10-5 MOE 25 46 32 94 12.5 0 0 5 0 0 Plasma Chemistry Markers

To evaluate the effect of ISIS oligonucleotides on day 8, plasma levels of transaminases (ALT and AST) were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.). The results are presented in the Table below. ISIS oligonucleotides that caused changes in the levels of any of these liver function markers outside the expected range for antisense oligonucleotides were excluded in further studies.

TABLE 194 Plasma transaminase levels (IU/L) in transgenic mice on day 8 Dose SEQ ID Chemistry (mg/kg) ALT AST NO PBS — — 22 82 ISIS 5-10-5 MOE 25 21 41 233 233710 12.5 23 66 5 22 118 ISIS 5-10-5 MOE 25 25 47 20 544199 12.5 20 40 5 27 43 ISIS Deoxy, MOE 25 21 34 110 559277 and cEt 12.5 21 37 2 22 39 ISIS 5-10-5 MOE 25 21 37 35 560400 12.5 20 44 5 24 35 ISIS Deoxy, MOE 25 22 48 113 561010 and cEt 12.5 33 64 5 24 41 ISIS 5-10-5 MOE 25 21 36 77 563580 12.5 29 81 5 21 59 ISIS 5-10-5 MOE 25 22 47 93 567320 12.5 29 58 5 21 70 ISIS 5-10-5 MOE 25 20 50 94 567321 12.5 24 102 5 19 53

Example 127: Tolerability of Antisense Oligonucleotides Targeting Human ANGPTL3 in CD1 Mice

CD1® mice (Charles River, Mass.) are a multipurpose mice model, frequently utilized for safety and efficacy testing. The mice were treated with ISIS antisense oligonucleotides selected from studies described above and evaluated for changes in the levels of various plasma chemistry markers.

Study 1

Male CD1 mice (one animal per treatment group) were injected intraperitoneally with a single dose of 200 mg/kg of deoxy, MOE, and cEt oligonucleotide. One male CD1 mouse was injected subcutaneously with a single dose of PBS. Mice were euthanized 48 hours after the last dose, and organs and plasma were harvested for further analysis.

Plasma Chemistry Markers

To evaluate the effect of ISIS oligonucleotides on day 4 plasma levels of transaminases (ALT and AST) were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.). The results are presented in the Table below. ISIS oligonucleotides that caused changes in the levels of any of these liver function markers outside the expected range for antisense oligonucleotides were excluded in further studies.

TABLE 195 Plasma transaminase levels in CD1 mice plasma on day 4 SEQ ALT AST ID (IU/L) (IU/L) NO ISIS 559277 29 43 110 ISIS 560990 19 43 111 ISIS 560992 21 36 112 ISIS 561010 31 40 113 ISIS 561011 27 32 114 ISIS 561022 35 48 115 ISIS 561025 17 28 116 ISIS 561026 31 43 117 ISIS 561208 32 47 118 ISIS 561320 25 37 119 ISIS 561343 41 90 120 ISIS 561345 30 45 121 ISIS 561347 31 41 122 ISIS 561458 18 38 124 ISIS 561460 42 59 125 ISIS 561463 21 33 127 ISIS 561486 17 39 130 ISIS 561487 18 39 131 ISIS 561504 24 41 133 ISIS 561621 31 56 134 Body Weights

Body weights were measured one day after the single dose of ISIS oligonucleotide, and are presented in the Table below. ISIS oligonucleotides that caused any changes in organ weights outside the expected range for antisense oligonucleotides were excluded from further studies.

TABLE 196 Body weights (g) of CD1 mice after antisense oligonucleotide treatment SEQ ID Body weight NO ISIS 559277 27 110 ISIS 560990 28 111 ISIS 560992 29 112 ISIS 561010 30 113 ISIS 561011 27 114 ISIS 561022 24 115 ISIS 561025 28 116 ISIS 561026 27 117 ISIS 561208 29 118 ISIS 561320 27 119 ISIS 561343 24 120 ISIS 561345 25 121 ISIS 561347 28 122 ISIS 561458 25 124 ISIS 561460 26 125 ISIS 561463 26 127 ISIS 561486 26 130 ISIS 561487 27 131 ISIS 561504 26 133 ISIS 561621 27 134 Study 2 Male CD1 mice (one animal per treatment group) were injected intraperitoneally with a single dose of 200 mg/kg of deoxy, MOE and cEt oligonucleotides. One male CD1 mouse was injected subcutaneously with a single dose of PBS. Mice were euthanized 48 hours after the last dose, and organs and plasma were harvested for further analysis. Plasma Chemistry Markers

To evaluate the effect of ISIS oligonucleotides on day 5 plasma levels of transaminases (ALT and AST) were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.). The results are presented in the Table below. ISIS oligonucleotides that caused changes in the levels of any of these liver function markers outside the expected range for antisense oligonucleotides were excluded in further studies.

TABLE 197 Plasma transaminase levels in CD1 mice plasma on day 5 SEQ ALT AST ID (IU/L) (IU/L) NO ISIS 561622 29 64 136 ISIS 561628 17 24 137 ISIS 561646 16 34 140 ISIS 561650 32 51 142 ISIS 561079 19 32 160 ISIS 561084 24 56 161 ISIS 561241 60 70 164 ISIS 561462 22 54 126 ISIS 561649 56 53 141 ISIS 561770 23 39 143 ISIS 561781 20 41 144 ISIS 561918 31 112 146 ISIS 562078 15 33 147 ISIS 562086 19 32 148 ISIS 562110 20 41 150 ISIS 562415 13 30 154 ISIS 562433 19 35 155 ISIS 562436 21 37 156 ISIS 562442 19 34 158 Body Weights

Body weights were measured on day 5 after the single dose of ISIS oligonucleotide, and are presented in the Table below. ISIS oligonucleotides that caused any changes in organ weights outside the expected range for antisense oligonucleotides were excluded from further studies.

TABLE 198 Body weights (g) of CD1 mice after antisense oligonucleotide treatment Body SEQ weights ID NO ISIS 561622 27 136 ISIS 561628 28 137 ISIS 561646 29 140 ISIS 561650 30 142 ISIS 561079 27 160 ISIS 561084 24 161 ISIS 561241 28 164 ISIS 561462 27 126 ISIS 561649 29 141 ISIS 561770 27 143 ISIS 561781 24 144 ISIS 561918 25 146 ISIS 562078 28 147 ISIS 562086 25 148 ISIS 562110 26 150 ISIS 562415 26 154 ISIS 562433 26 155 ISIS 562436 27 156 ISIS 562442 26 158 Study 3

Male CD1 mice (four animals per treatment group) were injected intraperitoneally with 100 mg/kg of 5-10-5 MOE gapmers given once a week for 6 weeks. One group of 4 male CD1 mice was injected intraperitoneally with PBS given once a week for 6 weeks. Mice were euthanized 48 hours after the last dose, and organs and plasma were harvested for further analysis.

Plasma Chemistry Markers

To evaluate the effect of ISIS oligonucleotides, plasma levels of various liver and kidney function markers were measured on day 45 using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.). The results are presented in the Table below. ISIS oligonucleotides that caused changes in the levels of any of these markers outside the expected range for antisense oligonucleotides were excluded in further studies.

TABLE 199 Plasma chemistry marker levels in CD1 mice plasma on day 45 ALT AST Albumin BUN Creatinine Bilurubin SEQ (IU/L) (IU/L) (g/dL) (mg/dL) (mg/dL) (mg/dL) ID NO PBS 30 55 2.7 26 0.15 0.17 ISIS 544145 1146 1081 2.5 29 0.14 0.24 16 ISIS 544199 244 213 2.6 25 0.13 0.15 20 ISIS 560400 211 244 2.5 28 0.14 0.14 35 ISIS 560401 212 269 2.4 31 0.14 0.12 36 ISIS 560469 165 160 2.4 24 0.11 0.14 38 ISIS 567320 141 146 2.7 25 0.14 0.15 93 ISIS 567321 106 122 2.5 24 0.11 0.13 94 Body Weights

Body weights were measured on day 43, and are presented in the Table below. Kidney, liver and spleen weights were measured at the end of the study on day 45. ISIS oligonucleotides that caused any changes in organ weights outside the expected range for antisense oligonucleotides were excluded from further studies.

TABLE 200 Weights (g) of CD1 mice after antisense oligonucleotide treatment SEQ ID Body Kidney Liver Spleen NO PBS 39 0.6 2.1 0.1 ISIS 544145 30 0.5 1.9 0.1 16 ISIS 544199 42 0.6 2.9 0.3 20 ISIS 560400 40 0.6 2.8 0.3 35 ISIS 560401 38 0.6 2.7 0.2 36 ISIS 560469 40 0.6 2.7 0.2 38 ISIS 567320 39 0.6 2.3 0.3 93 ISIS 567321 42 0.6 2.6 0.3 94 Study 4

Male CD1 mice (four animals per treatment group) were injected intraperitoneally with 50 mg/kg or 100 mg/kg of 5-10-5 MOE gapmers or deoxy, MOE and cEt oligonucleotides given once a week for 6 weeks. One group of 4 male CD1 mice was injected intraperitoneally with PBS given once a week for 6 weeks. Mice were euthanized 48 hours after the last dose, and organs and plasma were harvested for further analysis.

Plasma Chemistry Markers

To evaluate the effect of ISIS oligonucleotides, plasma levels of various liver and kidney function markers were measured on day 46 using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.). The results are presented in the Table below. ISIS oligonucleotides that caused changes in the levels of any of these markers outside the expected range for antisense oligonucleotides were excluded in further studies.

TABLE 201 Plasma chemistry marker levels in CD1 mice plasma on day 45 Dose ALT AST Albumin BUN Creatinine Bilurubin SEQ Chemistry (mg/kg) (IU/L) (IU/L) (g/dL) (mg/dL) (mg/dL) (mg/dL) ID NO PBS — 28 46 2.7 28 0.13 0.13 ISIS 544156 5-10-5 MOE 100 80 145 2.2 26 0.12 0.10 17 ISIS 560574 5-10-5 MOE 100 182 184 2.5 25 0.14 0.15 44 ISIS 561010 Deoxy, MOE 50 32 53 2.4 31 0.15 0.12 113 and cEt ISIS 561011 Deoxy, MOE 50 93 152 1.8 27 0.15 0.08 114 and cEt ISIS 560580 5-10-5 MOE 100 50 76 2.5 25 0.12 0.13 237 ISIS 567115 5-10-5 MOE 100 202 304 2.5 19 0.14 0.12 88 ISIS 567233 5-10-5 MOE 100 123 145 2.5 24 0.12 0.12 90 Body Weights

Body weights were measured on day 44, and are presented in the Table below. Kidney, liver and spleen weights were measured at the end of the study on day 46. ISIS oligonucleotides that caused any changes in organ weights outside the expected range for antisense oligonucleotides were excluded from further studies.

TABLE 202 Weights (g) of CD1 mice after antisense oligonucleotide treatment Dose SEQ Chemistry (mg/kg) Body Kidney Liver Spleen ID NO PBS — 38 0.6 2.1 0.2 ISIS 544156 5-10-5 MOE 100 36 0.5 2.2 0.2 17 ISIS 560574 5-10-5 MOE 100 40 0.6 2.6 0.4 44 ISIS 561010 Deoxy, MOE 50 39 0.5 2.2 0.2 113 and cEt ISIS 561011 Deoxy, MOE 50 39 0.6 2.9 0.3 114 and cEt ISIS 560580 5-10-5 MOE 100 39 0.5 2.4 0.2 237 ISIS 567115 5-10-5 MOE 100 36 0.5 2.2 0.2 88 ISIS 567233 5-10-5 MOE 100 39 0.6 2.2 0.3 90 Study 5

Male CD1 mice (four animals per treatment group) were injected intraperitoneally with 50 mg/kg of deoxy, MOE and cEt oligonucleotides given once a week for 6 weeks. One group of 4 male CD1 mice was injected intraperitoneally with PBS given once a week for 6 weeks. Mice were euthanized 48 hours after the last dose, and organs and plasma were harvested for further analysis.

Plasma Chemistry Markers

To evaluate the effect of ISIS oligonucleotides, plasma levels of various liver and kidney function markers were measured on day 43 using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.). The results are presented in the Table below. ISIS oligonucleotides that caused changes in the levels of any of these markers outside the expected range for antisense oligonucleotides were excluded in further studies.

TABLE 203 Plasma chemistry marker levels in CD1 mice plasma on day 43 ALT AST Albumin BUN Creatinine Bilurubin SEQ (IU/L) (IU/L) (g/dL) (mg/dL) (mg/dL) (mg/dL) ID NO PBS 35 166 2.6 29 0.12 0.32 ISIS 559277 45 77 2.5 29 0.13 0.16 110 ISIS 561022 826 802 2.9 29 0.13 0.99 115 ISIS 561025 146 183 2.3 28 0.14 0.13 116 ISIS 561026 93 154 2.6 26 0.11 0.16 117 ISIS 561079 1943 1511 2.9 28 0.15 0.94 160 ISIS 561084 153 227 2.6 27 0.12 0.16 161 ISIS 561123 49 90 2.5 31 0.13 0.13 163 ISIS 561436 29 57 2.6 25 0.12 0.12 170 Body Weights

Body weights were measured on day 41, and are presented in the Table below. Kidney, liver and spleen weights were measured at the end of the study on day 43. ISIS oligonucleotides that caused any changes in organ weights outside the expected range for antisense oligonucleotides were excluded from further studies.

TABLE 204 Weights (g) of CD1 mice after antisense oligonucleotide treatment SEQ ID Body Kidney Liver Spleen NO PBS 37 0.5 2.0 0.1 ISIS 559277 38 0.6 2.5 0.3 110 ISIS 561022 31 0.4 3.2 0.1 115 ISIS 561025 37 0.5 2.6 0.2 116 ISIS 561026 39 0.6 2.1 0.2 117 ISIS 561079 42 0.6 4.0 0.2 160 ISIS 561084 37 0.6 2.4 0.2 161 ISIS 561123 36 0.6 2.2 0.2 163 ISIS 561436 41 0.6 2.4 0.2 170

Example 128: Measurement of Viscosity of ISIS Antisense Oligonucleotides Targeting Human ANGPTL3

The viscosity of select antisense oligonucleotides from the studies described above was measured with the aim of screening out antisense oligonucleotides which have a viscosity of more than 40 centipoise (cP). Oligonucleotides having a viscosity greater than 40 cP would have less than optimal viscosity.

ISIS oligonucleotides (32-35 mg) were weighed into a glass vial, 120 μL of water was added and the antisense oligonucleotide was dissolved into solution by heating the vial at 50° C. Part (75 μL) of the pre-heated sample was pipetted to a micro-viscometer (Cambridge). The temperature of the micro-viscometer was set to 25° C. and the viscosity of the sample was measured. Another part (20 μL) of the pre-heated sample was pipetted into 10 mL of water for UV reading at 260 nM at 85° C. (Cary UV instrument). The results are presented in the Table below, where the concentration of each antisense oligonucleotide was 350 mg/ml, and indicate that most of the antisense oligonucleotides solutions are optimal in their viscosity under the criterion stated above.

TABLE 205 Viscosity of ISIS antisense oligonucleotides targeting human ANGPTL3 ISIS No. Viscosity (cP) SEQ ID NO 233710 14.65 233 337478 13.34 235 544145 11.97 16 544162 8.50 18 544199 11.70 20 560306 5.67 34 560400 9.26 35 560401 18.11 36 560402 90.67 37 560469 12.04 38 560735 7.49 49 567320 9.05 93 567321 9.62 94 567233 6.72 90 563580 16.83 77 561010 26.32 113 561011 43.15 114

Example 129: Tolerability of Antisense Oligonucleotides Targeting Human ANGPTL3 in Sprague-Dawley Rats

Sprague-Dawley rats are a multipurpose model used for safety and efficacy evaluations. The rats were treated with ISIS antisense oligonucleotides from the studies described in the Examples above and evaluated for changes in the levels of various plasma chemistry markers.

Study 1

Male Sprague-Dawley rats were maintained on a 12-hour light/dark cycle and fed ad libitum with Purina normal rat chow, diet 5001. Groups of 4 Sprague-Dawley rats each were injected subcutaneously once a week for 6 weeks with PBS or with 100 mg/kg of 5-10-5 MOE gapmers. Forty eight hours after the last dose, rats were euthanized and organs and plasma were harvested for further analysis.

Liver Function

To evaluate the effect of ISIS oligonucleotides on hepatic function, plasma levels of transaminases were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.). Plasma levels of ALT (alanine transaminase) and AST (aspartate transaminase) were measured on day 45 and the results are presented in the Table below expressed in IU/L. Plasma levels of bilirubin were also measured using the same clinical chemistry analyzer and the results are also presented in the Table below expressed in mg/dL. ISIS oligonucleotides that caused changes in the levels of any markers of liver function outside the expected range for antisense oligonucleotides were excluded in further studies.

TABLE 206 Liver function markers in Sprague-Dawley rats ALT AST Bilirubin SEQ ID (IU/L) (IU/L) (mg/dL) NO PBS 25 65 0.11 ISIS 544145 225 407 0.30 16 ISIS 544199 56 102 0.11 20 ISIS 560400 55 175 0.12 35 ISIS 560401 89 206 0.13 36 ISIS 560469 227 290 0.15 38 ISIS 567320 55 172 0.11 93 ISIS 567321 39 109 0.10 94 Kidney Function

To evaluate the effect of ISIS oligonucleotides on kidney function, plasma levels of blood urea nitrogen (BUN) and creatinine were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.). Results are presented in the Table below, expressed in mg/dL. ISIS oligonucleotides that caused changes in the levels of any of the kidney function markers outside the expected range for antisense oligonucleotides were excluded in further studies. Total urine protein and urine creatinine levels were measured, and the ratio of total urine protein to creatinine was evaluated. The results are presented in the Table below.

TABLE 207 Kidney function plasma markers (mg/dL) in Sprague-Dawley rats SEQ ID BUN Creatinine NO PBS 16 0.27 ISIS 544145 53 0.26 16 ISIS 544199 24 0.34 20 ISIS 560400 28 0.31 35 ISIS 560401 29 0.28 36 ISIS 560469 23 0.32 38 ISIS 567320 26 0.35 93 ISIS 567321 24 0.37 94

TABLE 208 Kidney function urine markers in Sprague-Dawley rats Total Creatinine protein Protein:Creatinine SEQ ID (mg/dL) (mg/dL) ratio NO PBS 59 90 1.5 ISIS 544145 27 2131 84.8 16 ISIS 544199 24 199 8.6 20 ISIS 560400 32 176 5.4 35 ISIS 560401 29 521 17.3 36 ISIS 560469 43 351 8.2 38 ISIS 567320 34 177 5.2 93 ISIS 567321 54 269 5.3 94 Organ Weights

Body weights were measured on day 42 and presented in the Table below. Liver, spleen and kidney weights were measured at the end of the study on day 45, and are presented in the Table below. ISIS oligonucleotides that caused any changes in organ weights outside the expected range for antisense oligonucleotides were excluded from further studies.

TABLE 209 Body and organ weights (g) of Sprague Dawley rats SEQ ID Body Kidney Liver Spleen NO PBS 441 3.3 11.8 0.8 ISIS 544145 240 3.0 11.2 1.7 16 ISIS 544199 307 2.6 10.3 2.0 20 ISIS 560400 294 2.8 12.3 2.0 35 ISIS 560401 281 3.4 11.6 2.3 36 ISIS 560469 316 3.0 11.8 2.0 38 ISIS 567320 312 3.1 12.4 2.5 93 ISIS 567321 332 3.3 11.6 2.3 94 Study 2

Male Sprague-Dawley rats were maintained on a 12-hour light/dark cycle and fed ad libitum with Purina normal rat chow, diet 5001. Groups of 4 Sprague-Dawley rats each were injected subcutaneously once a week for 6 weeks with PBS or with 50 mg/kg or 100 mg/kg of 5-10-5 MOE gapmers or deoxy, MOE and cEt oligonucleotides. Forty eight hours after the last dose, rats were euthanized and organs and plasma were harvested for further analysis.

Liver Function

To evaluate the effect of ISIS oligonucleotides on hepatic function, plasma levels of transaminases were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.). Plasma levels of ALT (alanine transaminase) and AST (aspartate transaminase) were measured on day 44 and the results are presented in the Table below expressed in IU/L. Plasma levels of bilirubin were also measured using the same clinical chemistry analyzer and the results are also presented in the Table below expressed in mg/dL. ISIS oligonucleotides that caused changes in the levels of any markers of liver function outside the expected range for antisense oligonucleotides were excluded in further studies.

TABLE 210 Liver function markers in Sprague-Dawley rats Dose ALT AST Bilirubin SEQ Chemistry (mg/kg) (IU/L) (IU/L) (mg/dL) ID NO PBS — — 22 63 0.09 ISIS 544156 5-10-5 MOE 100 153 221 0.19 17 ISIS 560574 5-10-5 MOE 100 62 128 0.24 44 ISIS 561010 Deoxy, MOE 50 32 99 0.12 113 and cEt ISIS 561011 Deoxy, MOE 50 56 100 0.11 114 and cEt ISIS 563580 5-10-5 MOE 100 74 89 0.09 77 ISIS 567233 5-10-5 MOE 100 41 136 0.08 90 Kidney Function

To evaluate the effect of ISIS oligonucleotides on kidney function, plasma levels of blood urea nitrogen (BUN) and creatinine were measured on day 44 using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.). Results are presented in the Table below, expressed in mg/dL. ISIS oligonucleotides that caused changes in the levels of any of the kidney function markers outside the expected range for antisense oligonucleotides were excluded in further studies. Total urine protein and urine creatinine levels were measured, and the ratio of total urine protein to creatinine was evaluated. The results are presented in the Table below.

TABLE 211 Kidney function plasma markers (mg/dL) in Sprague-Dawley rats Dose SEQ ID Chemistry (mg/kg) BUN Creatinine NO PBS — — 18 0.31 ISIS 544156 5-10-5 MOE 100 27 0.27 17 ISIS 560574 5-10-5 MOE 100 32 0.24 44 ISIS 561010 Deoxy, MOE and 50 24 0.31 113 cEt ISIS 561011 Deoxy, MOE and 50 33 0.32 114 cEt ISIS 563580 5-10-5 MOE 100 25 0.20 77 ISIS 567233 5-10-5 MOE 100 37 0.23 90

TABLE 212 Kidney function urine markers in Sprague-Dawley rats Total Protein: Dose Creatinine protein Creatinine SEQ Chemistry (mg/kg) (mg/dL) (mg/dL) ratio ID NO PBS — — 55 66 1.2 ISIS 544156 5-10-5 MOE 100 26 166 6.2 17 ISIS 560574 5-10-5 MOE 100 39 276 6.8 44 ISIS 561010 Deoxy, MOE 50 54 299 5.6 113 and cEt ISIS 561011 Deoxy, MOE 50 41 525 11.7 114 and cEt ISIS 563580 5-10-5 MOE 100 44 338 8.1 77 ISIS 567233 5-10-5 MOE 100 46 307 6.4 90

Body weights were measured on day 42 and presented in the Table below. Liver, spleen and kidney weights were measured at the end of the study on day 44, and are presented in the Table below. ISIS oligonucleotides that caused any changes in organ weights outside the expected range for antisense oligonucleotides were excluded from further studies.

TABLE 213 Body and organ weights (g) of Sprague Dawley rats Dose SEQ Chemistry (mg/kg) Body Kidney Liver Spleen ID NO PBS — — 433 3.1 10.8 0.6 ISIS 544156 5-10-5 MOE 100 291 2.4 10.6 1.6 17 ISIS 560574 5-10-5 MOE 100 315 3.1 10.7 2.1 44 ISIS 561010 Deoxy, MOE 50 386 3.0 11.9 2.1 113 and cEt ISIS 561011 Deoxy, MOE 50 324 4.1 12.5 2.4 114 and cEt ISIS 563580 5-10-5 MOE 100 358 3.0 12.8 1.5 77 ISIS 567233 5-10-5 MOE 100 286 2.9 13.0 2.9 90

Study 3

Male Sprague-Dawley rats were maintained on a 12-hour light/dark cycle and fed ad libitum with Purina normal rat chow, diet 5001. Groups of 4 Sprague-Dawley rats each were injected subcutaneously once a week for 6 weeks with PBS or with 50 mg/kg of deoxy, MOE and cEt oligonucleotides. Forty eight hours after the last dose, rats were euthanized and organs and plasma were harvested for further analysis.

Liver Function

To evaluate the effect of ISIS oligonucleotides on hepatic function, plasma levels of transaminases were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.). Plasma levels of ALT (alanine transaminase) and AST (aspartate transaminase) were measured on day 44 and the results are presented in the Table below expressed in IU/L. Plasma levels of bilirubin were also measured using the same clinical chemistry analyzer and the results are also presented in the Table below expressed in mg/dL. ISIS oligonucleotides that caused changes in the levels of any markers of liver function outside the expected range for antisense oligonucleotides were excluded in further studies.

TABLE 214 Liver function markers in Sprague-Dawley rats ALT AST Bilirubin SEQ ID (IU/L) (IU/L) (mg/dL) NO PBS 27 87 0.08 ISIS 559277 36 108 0.10 110 ISIS 561025 150 260 0.15 116 ISIS 561026 53 105 0.08 117 ISIS 561079 87 196 0.09 160 ISIS 561084 62 177 0.11 161 ISIS 561123 39 94 0.07 163 ISIS 561436 64 225 0.13 170 Kidney Function

To evaluate the effect of ISIS oligonucleotides on kidney function, plasma levels of blood urea nitrogen (BUN) and creatinine were measured on day 44 using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.). Results are presented in the Table below, expressed in mg/dL. ISIS oligonucleotides that caused changes in the levels of any of the kidney function markers outside the expected range for antisense oligonucleotides were excluded in further studies. Total urine protein and urine creatinine levels were measured, and the ratio of total urine protein to creatinine was evaluated. The results are presented in the Table below.

TABLE 215 Kidney function plasma markers (mg/dL) in Sprague-Dawley rats SEQ ID BUN Creatinine NO PBS 12 0.26 ISIS 559277 16 0.30 110 ISIS 561025 24 0.34 116 ISIS 561026 61 0.38 117 ISIS 561079 87 0.67 160 ISIS 561084 24 0.35 161 ISIS 561123 16 0.31 163 ISIS 561436 39 0.37 170

TABLE 216 Kidney function urine markers in Sprague-Dawley rats Total Creatinine protein Protein:Creatinine SEQ ID (mg/dL) (mg/dL) ratio NO PBS 42 77 1.9 ISIS 559277 35 253 7.2 110 ISIS 561025 47 583 14.3 116 ISIS 561026 22 1993 111.4 117 ISIS 561079 17 1313 75.5 160 ISIS 561084 73 571 7.9 161 ISIS 561123 33 925 29.5 163 ISIS 561436 25 789 36.6 170 Organ Weights

Body weights were measured on day 42 and presented in the table below. Liver, spleen and kidney weights were measured at the end of the study on day 44, and are presented in the Table below. ISIS oligonucleotides that caused any changes in organ weights outside the expected range for antisense oligonucleotides were excluded from further studies.

TABLE 217 Body and organ weights (g) of Sprague Dawley rats SEQ ID Body Kidney Liver Spleen NO PBS 419 3.2 10.7 0.7 ISIS 559277 365 3.5 11.2 1.6 110 ISIS 561025 335 3.2 12.8 2.7 116 ISIS 561026 334 4.9 13.9 2.3 117 ISIS 561079 302 3.9 9.9 0.9 160 ISIS 561084 317 3.5 12.2 1.9 161 ISIS 561123 367 3.3 13.5 1.5 163 ISIS 561436 272 3.1 9.8 2.9 170

Example 130: Effect of ISIS Antisense Oligonucleotides Targeting Human ANGPTL3 in Cynomolgus Monkeys

Cynomolgus monkeys were treated with ISIS antisense oligonucleotides selected from studies described in the Examples above. Antisense oligonucleotide efficacy and tolerability, as well as their pharmacokinetic profile in the liver and kidney, were evaluated.

At the time this study was undertaken, the cynomolgus monkey genomic sequence was not available in the National Center for Biotechnology Information (NCBI) database; therefore, cross-reactivity with the cynomolgus monkey gene sequence could not be confirmed. Instead, the sequences of the ISIS antisense oligonucleotides used in the cynomolgus monkeys was compared to a rhesus monkey sequence for homology. It is expected that ISIS oligonucleotides with homology to the rhesus monkey sequence are fully cross-reactive with the cynomolgus monkey sequence as well. The human antisense oligonucleotides tested are cross-reactive with the rhesus genomic sequence (GENBANK Accession No. NW_001108682.1 truncated from nucleotides 3049001 to 3062000, designated herein as SEQ ID NO: 3). The greater the complementarity between the human oligonucleotide and the rhesus monkey sequence, the more likely the human oligonucleotide can cross-react with the rhesus monkey sequence. The start and stop sites of each oligonucleotide to SEQ ID NO: 3 is presented in the Table below. “Start site” indicates the 5′-most nucleotide to which the gapmer is targeted in the rhesus monkey gene sequence. ‘Mismatches’ indicates the number of nucleobases in the human oligonucleotide that are mismatched with the rhesus genomic sequence.

TABLE 218 Antisense oligonucleotides complementary to the rhesus ANGPTL3 genomic sequence (SEQ ID NO: 3) Target SEQ Start ID ISIS No Site Mismatches Chemistry NO 563580 9315 2 5-10-5 MOE 77 560400 10052 1 5-10-5 MOE 35 567320 10232 1 5-10-5 MOE 93 567321 10234 1 5-10-5 MOE 94 544199 10653 0 5-10-5 MOE 20 567233 6834 2 5-10-5 MOE 90 561011 3220 1 Deoxy, MOE and (S)-cEt 114 559277 3265 0 Deoxy, MOE and (S)-cEt 110 Treatment

Prior to the study, the monkeys were kept in quarantine for at least a 30 day period, during which the animals were observed daily for general health. The monkeys were 2-4 years old and weighed between 2 and 4 kg. Nine groups of 5 randomly assigned male cynomolgus monkeys each were injected subcutaneously with ISIS oligonucleotide or PBS at four sites on the back in a clockwise rotation (i.e. left, top, right, and bottom), one site per dose. The monkeys were given loading doses of PBS or 40 mg/kg of ISIS oligonucleotide every two days for the first week (days 1, 3, 5, and 7) and were subsequently dosed once a week for 12 weeks (days 14, 21, 28, 35, 42, 49, 56, 63, 70, 77, and 84) with PBS or 40 mg/kg of ISIS oligonucleotide.

During the study period, the monkeys were observed twice daily for signs of illness or distress. Any animal experiencing more than momentary or slight pain or distress due to the treatment, injury or illness was treated by the veterinary staff with approved analgesics or agents to relieve the pain after consultation with the Study Director. Any animal in poor health or in a possible moribund condition was identified for further monitoring and possible euthanasia. For example, one animal in the ISIS 567321 treatment group was found moribund on day 45 and was terminated. Scheduled euthanasia of the animals was conducted on day 86 (approximately 48 hours after the final dose) by exsanguination after ketamine/xylazine-induced anesthesia and administration of sodium pentobarbital. The protocols described in the Example were approved by the Institutional Animal Care and Use Committee (IACUC).

Hepatic Target Reduction

RNA Analysis

On day 86, RNA was extracted from liver for real-time PCR analysis of measurement of mRNA expression of ANGPTL3. Results are presented as percent change of mRNA, relative to PBS control, normalized with RIBOGREEN®. As shown in the Table below, treatment with ISIS antisense oligonucleotides resulted in significant reduction of ANGPTL3 mRNA in comparison to the PBS control.

Analysis of ANGPTL3 mRNA levels revealed that ISIS 544199 and ISIS 559277, which are both fully cross-reactive with the rhesus sequence, significantly reduced expression levels. Other ISIS oligonucleotides, which targeted the monkey sequence with mismatches, were also able to reduce ANGPTL3 mRNA levels.

TABLE 219 Percent inhibition of ANGPTL3 mRNA in the cynomolgus monkey liver relative to the PBS control SEQ ISIS No % ID NO 563580 62 77 560400 59 35 567320 67 93 567321 34 94 544199 88 20 561011 47 114 559277 85 110 Protein Analysis

Approximately 1 mL of blood was collected from all available animals at day 85 and placed in tubes containing the potassium salt of EDTA. The blood samples were placed in ice and centrifuged (3000 rpm for 10 min at 4° C.) to obtain plasma.

Human ANGPTL3 protein levels were quantified using a commercially available ELISA kit (Catalog #DANL30 by R&D Systems, Minneapolis, Minn.) with transgenic plasma samples diluted 1:20,000 using the manufacturer described protocol. The results are presented in the Table below. Analysis of plasman ANGPTL3 revealed that ISIS 563580, 544199 and ISIS 559277 reduced protein levels in a sustained manner. Other ISIS oligonucleotides were also able to reduce ANGPTL3 levels.

TABLE 220 Plasma protein levels (ng/mL) in the cynomolgus monkey SEQ Day 1 Day 3 Day 16 Day 30 Day 44 Day 58 Day 72 Day 86 ID NO PBS 142 113 122 75 147 170 130 158 ISIS 563580 113 99 102 46 109 93 82 81 77 ISIS 560400 92 107 145 63 170 182 157 178 35 ISIS 567320 87 72 94 56 176 181 134 166 93 ISIS 567321 80 84 98 62 156 116 122 112 94 ISIS 544199 114 84 50 34 66 56 81 71 20 ISIS 567233 115 111 174 134 162 125 122 109 90 ISIS 561011 89 92 111 106 104 100 140 129 114 ISIS 559277 86 62 63 54 77 64 68 70 110 Tolerability Studies Body Weight Measurements

To evaluate the effect of ISIS oligonucleotides on the overall health of the animals, body and weights were measured and are presented in the Table below. The results indicate that effect of treatment with antisense oligonucleotides on body weights was within the expected range for antisense oligonucleotides. Specifically, treatment with ISIS 563580 was well tolerated in terms of the body weights of the monkeys.

TABLE 221 Final body weights (g) in cynomolgus monkey SEQ Day 1 Day 14 Day 28 Day 35 Day 56 Day 70 Day 84 ID NO PBS 2713 2709 2721 2712 2761 2754 2779 ISIS 563580 2678 2669 2724 2699 2797 2798 2817 77 ISIS 560400 2713 2738 2808 2767 2867 2920 2976 35 ISIS 567320 2682 2707 2741 2731 2804 2830 2853 93 ISIS 567321 2672 2745 2849 2845 2995 2965 3002 94 ISIS 544199 2760 2813 2851 2897 2905 2888 2871 20 ISIS 567233 2657 2668 2650 2677 2907 2963 2903 90 ISIS 561011 2753 2797 2801 2811 2921 2967 2941 114 ISIS 559277 2681 2688 2701 2755 2826 2831 2965 110 Liver Function

To evaluate the effect of ISIS oligonucleotides on hepatic function, blood samples were collected from all the study groups. The blood samples were collected from the cephalic, saphenous, or femoral veins, 48 hours post-dosing. The monkeys were fasted overnight prior to blood collection. Blood was collected in tubes without anticoagulant for serum separation. The tubes were kept at room temperature for a minimum of 90 minutes and then centrifuged (approximately 3,000 rpm for 10 min) to obtain serum. Levels of various liver function markers were measured using a Toshiba 200FR NEO chemistry analyzer (Toshiba Co., Japan). Plasma levels of ALT and AST were measured and the results are presented in the Table below, expressed in IU/L. Bilirubin, a liver function marker, was similarly measured and is presented in the Table below expressed in mg/dL. The results indicate that most of the antisense oligonucleotides had no effect on liver function outside the expected range for antisense oligonucleotides. Specifically, treatment with ISIS 563580 was well tolerated in terms of the liver function in monkeys.

TABLE 222 ALT levels (IU/L) in cynomolgus monkey plasma SEQ Day 1 Day 30 Day 58 Day 86 ID NO PBS 47 35 32 46 ISIS 563580 56 55 55 83 77 ISIS 560400 50 35 47 68 35 ISIS 567320 72 44 51 106 93 ISIS 567321 53 39 44 75 94 ISIS 544199 58 49 51 51 20 ISIS 567233 42 38 47 64 90 ISIS 561011 48 35 34 43 114 ISIS 559277 49 45 53 60 110

TABLE 223 AST levels (IU/L) in cynomolgus monkey plasma SEQ Day 1 Day 30 Day 58 Day 86 ID NO PBS 76 42 39 60 ISIS 563580 75 56 42 81 77 ISIS 560400 85 63 59 99 35 ISIS 567320 104 64 55 153 93 ISIS 567321 83 47 45 66 94 ISIS 544199 68 68 70 91 20 ISIS 567233 46 80 66 86 90 ISIS 561011 48 39 41 51 114 ISIS 559277 50 56 55 77 110

TABLE 224 Bilirubin levels (mg/dL) in cynomolgus monkey plasma SEQ Day 1 Day 30 Day 58 Day 86 ID NO PBS 0.31 0.24 0.20 0.19 ISIS 563580 0.34 0.23 0.17 0.18 77 ISIS 560400 0.29 0.19 0.14 0.13 35 ISIS 567320 0.38 0.24 0.16 0.19 93 ISIS 567321 0.35 0.20 0.16 0.17 94 ISIS 544199 0.23 0.16 0.17 0.15 20 ISIS 567233 0.26 0.17 0.15 0.12 90 ISIS 561011 0.20 0.13 0.16 0.13 114 ISIS 559277 0.22 0.15 0.16 0.15 110 Kidney Function

To evaluate the effect of ISIS oligonucleotides on kidney function, blood samples were collected from all the study groups. The blood samples were collected from the cephalic, saphenous, or femoral veins, 48 hours post-dosing. The monkeys were fasted overnight prior to blood collection. Blood was collected in tubes without anticoagulant for serum separation. The tubes were kept at room temperature for a minimum of 90 minutes and then centrifuged (approximately 3,000 rpm for 10 min) to obtain serum. Levels of BUN and creatinine were measured using a Toshiba 200FR NEO chemistry analyzer (Toshiba Co., Japan). Results are presented in the Table below, expressed in mg/dL.

The plasma chemistry data indicate that most of the ISIS oligonucleotides did not have any effect on the kidney function outside the expected range for antisense oligonucleotides. Specifically, treatment with ISIS 563580 was well tolerated in terms of the kidney function of the monkeys.

TABLE 225 Plasma BUN levels (mg/dL) in cynomolgus monkeys SEQ Day 1 Day 30 Day 58 Day 86 ID NO PBS 28 28 27 29 ISIS 563580 27 27 25 27 77 ISIS 560400 25 24 21 27 35 ISIS 567320 27 28 26 32 93 ISIS 567321 25 24 23 24 94 ISIS 544199 23 25 24 23 20 ISIS 567233 23 32 30 29 90 ISIS 561011 25 24 23 24 114 ISIS 559277 26 28 24 26 110

TABLE 226 Plasma creatinine levels (mg/dL) in cynomolgus monkeys SEQ Day 1 Day 30 Day 58 Day 86 ID NO PBS 0.96 0.95 0.89 0.88 ISIS 563580 0.97 1.04 0.88 0.85 77 ISIS 560400 0.99 1.00 0.93 0.91 35 ISIS 567320 0.95 0.94 0.89 0.87 93 ISIS 567321 0.97 0.94 0.89 0.87 94 ISIS 544199 0.86 0.87 0.88 0.87 20 ISIS 567233 0.89 1.08 1.06 1.00 90 ISIS 561011 0.93 0.93 0.91 0.90 114 ISIS 559277 0.86 0.91 0.87 0.91 110 Hematology

To evaluate any effect of ISIS oligonucleotides in cynomolgus monkeys on hematologic parameters, blood samples of approximately 0.5 mL of blood was collected from each of the available study animals in tubes containing K₂-EDTA. Samples were analyzed for red blood cell (RBC) count, white blood cells (WBC) count, individual white blood cell counts, such as that of monocytes, neutrophils, lymphocytes, as well as for platelet count, hemoglobin content and hematocrit, using an ADVIA120 hematology analyzer (Bayer, USA). The data is presented in the Tables below.

The data indicate the oligonucleotides did not cause any changes in hematologic parameters outside the expected range for antisense oligonucleotides at this dose. Specifically, treatment with ISIS 563580 was well tolerated in terms of the hematologic parameters of the monkeys.

TABLE 227 Blood cell counts in cynomolgus monkeys RBC Platelets WBC Neutrophils Lymphocytes Monocytes SEQ (×10⁶/μL) (×10³/μL) (×10³/μL) (% WBC) (% total) (% total) ID NO PBS 5.6 462 12.2 58 39 2 ISIS 563580 5.5 394 10.7 52 44 2 77 ISIS 560400 5.7 269 10.2 44 50 3 35 ISIS 567320 5.1 329 9.1 51 44 3 93 ISIS 567321 5.3 363 8.9 60 36 2 94 ISIS 544199 5.6 316 9.7 34 61 3 20 ISIS 567233 5.0 298 12.1 40 53 4 90 ISIS 561011 5.5 356 10.2 33 62 3 114 ISIS 559277 5.1 343 8.3 45 49 3 110

TABLE 228 Hematologic parameters in cynomolgus monkeys Hemoglobin HCT SEQ (g/dL) (%) ID NO PBS 13 43 ISIS 563580 12 40 77 ISIS 560400 12 41 35 ISIS 567320 11 38 93 ISIS 567321 12 41 94 ISIS 544199 13 44 20 ISIS 567233 11 38 90 ISIS 561011 13 42 114 ISIS 559277 12 40 110 Effect on Pro-Inflammatory Molecules

To evaluate any inflammatory effect of ISIS oligonucleotides in cynomolgus monkeys, blood samples were taken for analysis of C-reactive protein and C3 levels on day 84 pre-dose. Approximately 1.5 mL of blood was collected from each animal and put into tubes without anticoagulant for serum separation. The tubes were kept at room temperature for a minimum of 90 min and then centrifuged at 3,000 rpm for 10 min at room temperature to obtain serum. C-reactive protein (CRP) and complement C3, which serve as markers of inflammation, were measured using a Toshiba 200FR NEO chemistry analyzer (Toshiba Co., Japan). The results indicate that treatment with ISIS 563580 was tolerable in monkeys.

TABLE 229 C-reactive protein levels (mg/L) in cynomolgus monkey plasma SEQ Day 1 Day 30 Day 58 Day 86 ID NO PBS 3.1 5.5 2.7 4.1 ISIS 563580 2.4 2.4 4.5 3.9 77 ISIS 560400 3.4 7.5 9.2 14.4 35 ISIS 567320 2.5 1.7 2.5 4.3 93 ISIS 567321 3.7 3.1 5.5 7.0 94 ISIS 544199 1.2 1.5 8.8 8.1 20 ISIS 567233 1.9 12.0 6.8 6.6 90 ISIS 561011 1.7 1.2 2.1 3.7 114 ISIS 559277 1.8 2.1 10.9 5.2 110

TABLE 230 C3 levels (mg/dL) in cynomolgus monkey plasma SEQ Pre-dose Day 84 ID NO PBS 122 117 ISIS 563580 116 84 77 ISIS 560400 120 105 35 ISIS 567320 114 100 93 ISIS 567321 106 93 94 ISIS 544199 113 66 20 ISIS 567233 113 63 90 ISIS 561011 115 79 114 ISIS 559277 119 87 110 Measurement of Oligonucleotide Concentration

The concentration of the full-length oligonucleotide was measured. The method used is a modification of previously published methods (Leeds et al., 1996; Geary et al., 1999) which consist of a phenol-chloroform (liquid-liquid) extraction followed by a solid phase extraction. An internal standard (ISIS 355868, a 27-mer 2′-O-methoxyethyl modified phosphorothioate oligonucleotide, GCGTTTGCTCTTCTTCTTGCGTTTTTT, designated herein as SEQ ID NO: 13) was added prior to extraction. Tissue sample concentrations were calculated using calibration curves, with a lower limit of quantitation (LLOQ) of approximately 1.14 μg/g. The results are presented in the Table below, expressed as g/g liver or kidney tissue. The ratio of full-length oligonucleotide concentrations in the kidney versus the liver was calculated. The ratio of full-length oligonucleotide concentrations in the kidney versus the liver after treatment with ISIS 563580 was found to be most optimal compared to other compounds assessed.

TABLE 231 Oligonucleotide full length concentration Kidney/Liver SEQ ISIS No Kidney Liver ratio ID NO 563580 1822 1039 1.8 77 560400 3807 1375 2.8 35 567320 2547 569 4.5 93 567321 2113 463 4.6 94 544199 1547 561 2.8 20 561011 2027 477 4.3 114 559277 2201 508 4.3 110

Example 131: Comparison of Antisense Inhibition of Human ANGPTL3 in huANGPTL3 Transgenic Mice by ISIS Oligonucleotides Comprising a GalNAc Conjugate Group and ISIS Oligonucleotides that do not Comprise a GalNAc Conjugate Group

Antisense oligonucleotides comprising GalNAc₃-7_(a) were evaluated in comparison with their unconjugated counterparts for their ability to reduce human ANGPTL3 mRNA transcript in Tg mice. The gapmers, which target SEQ ID NO: 1, are described in the Table below and in Table 121. The symbols of the Backbone Chemistry column are as follows: ‘s’ denotes thioate ester and ‘o’ denotes phosphate ester.

TABLE 232 ISIS oligonucleotides Target SEQ Start Backbone ID ISIS No Sequence Site Conjugate Chemistry NO 563580 GGACATTGCCAGTAATCGCA 1140 None sssssssssssssssssss 77 703801 GGACATTGCCAGTAATCGCA 1140 GalNAc₃-7a sssssssssssssssssss 77 703802 GGACATTGCCAGTAATCGCA 1140 GalNAc₃-7a soooossssssssssooss 77

Female and male Tg mice were maintained on a 12-hour light/dark cycle. Animals were acclimated for at least 7 days in the research facility before initiation of the experiment. Antisense oligonucleotides (ASOs) were prepared in buffered saline (PBS) and sterilized by filtering through a 0.2 micron filter. Oligonucleotides were dissolved in 0.9% PBS for injection.

A group of 4 mice received subcutaneous injections of ISIS 563580 at doses of 5 mg/kg, 10 mg/kg, 15 mg/kg, or 30 mg/kg once per week for 2 weeks. Groups of 4 mice each received intraperitoneal injections of ISIS 703801 or ISIS 703802 at doses of 0.3 mg/kg, 1 mg/kg, 3 mg/kg, or 10 mg/kg once per week for 2 weeks. One group of mice received subcutaneous injections of PBS once weekly for 2 weeks. The PBS-injected group served as the control group to which the corresponding oligonucleotide-treated groups were compared.

RNA Analysis

At the end of the treatment period, RNA was extracted from liver tissue for real-time PCR analysis of measurement of mRNA expression of ANGPTL3 with human primer probe set hANGPTL3_LTS01022. Results are presented as percent change of mRNA, relative to PBS control, normalized with RIBOGREEN®. A zero value simply indicates that the antisense oligonucleotide did not inhibit expression at a measurable level.

The results demonstrate that the conjugated compounds are much more potent in reducing ANGPTL3 expression than their unconjugated counterpart as evident from the percent inhibition and ID₅₀ values. The conjugated oligonucleotide with mixed backbone chemistry (703802) was more potent in inhibiting expression than the conjugated oligonucleotide with full phosphorothioate backbone chemistry (703801).

TABLE 233 Percent inhibition of ANGPTL3 mRNA in transgenic mouse liver relative to the PBS control Dose % ID₅₀ ISIS No (mg/kg) inhibition (mg/kg/wk) 563580 30 79 6 15 73 10 72 5 40 703801 10 85 1 3 89 1 54 0.3 32 703802 10 89 0.3 3 85 1 67 0.3 52 Protein Analysis

Human ANGPTL3 protein levels were quantified using a commercially available ELISA kit (Catalog #DANL30 by R&D Systems, Minneapolis, Minn.) with transgenic plasma samples diluted 1:20,000 using the manufacturer described protocol. The results are presented in the Table below. A zero value simply indicates that the antisense oligonucleotide did not inhibit expression at a measurable level.

The results demonstrate that the conjugated compounds are more potent in reducing ANGPTL3 expression than their unconjugated counterpart as evident from the percent inhibition values. The conjugated oligonucleotide with mixed backbone chemistry (703802) was more potent in inhibiting expression than the conjugated oligonucleotide with full phosphorothioate backbone chemistry (703801).

TABLE 234 Percent inhibition of plasma protein levels in the transgenic mouse Dose % ISIS No (mg/kg) inhibition 563580 30 77 15 74 10 75 5 56 703801 10 82 3 40 1 0 0.3 0 703802 10 81 3 81 1 64 0.3 66

Example 132: Tolerability of a GalNAc Conjugated Antisense Oligonucleotide Targeting Human ANGPTL3 in CD1 Mice

Male CD1 mice (four animals per treatment group) were injected subcutaneously with various doses of ISIS 703802 as described in the Table below for 6 weeks (on days 1, 3, 5, 8, 14, 21, 28, 35 and 42). One group of 4 male CD1 mice was injected subcutaneously with PBS for 6 weeks. Mice were euthanized 48 hours after the last dose, and organs and plasma were harvested for further analysis.

Plasma Chemistry Markers

To evaluate the effect of ISIS 703802, plasma levels of various liver and kidney function markers were measured on day 44 using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.). The results are presented in the Table below. ISIS 703802 was shown to be a tolerable compound even at high doses.

TABLE 235 Plasma chemistry marker levels in CD1 mice plasma on day 44 ALT AST Albumin BUN Creatinine Bilurubin (IU/L) (IU/L) (g/dL) (mg/dL) (mg/dL) (mg/dL) PBS 34 49 2.6 27 0.15 0.13 ISIS 703802 96 81 2.8 25 0.17 0.17 50 mg/kg/wk ISIS 703802 54 56 2.7 27 0.16 0.17 20 mg/kg/wk ISIS 703802 37 49 2.7 28 0.19 0.15 10 mg/kg/wk ISIS 703802 36 46 2.7 26 0.16 0.16  5 mg/kg/wk Body Weights

Body, kidney, liver and spleen weights were measured at the end of the study on day 44. ISIS 703802 did not significantly change body and organ weights even when administered at high doses.

TABLE 236 Weights (g) of CD1 mice after antisense oligonucleotide treatment Body Kidney Liver Spleen PBS 42 0.64 2.06 0.12 ISIS 703802 39 0.53 2.42 0.10 50 mg/kg/wk ISIS 703802 40 0.57 2.29 0.13 20 mg/kg/wk ISIS 703802 43 0.66 2.36 0.13 10 mg/kg/wk ISIS 703802 42 0.63 2.38 0.13  5 mg/kg/wk

Example 133: Tolerability of a GalNAc Conjugated Antisense Oligonucleotide Targeting Human ANGPTL3 in Sprague-Dawley Rats

Sprague-Dawley rats are a multipurpose model used for safety and efficacy evaluations. Male Sprague-Dawley rats were maintained on a 12-hour light/dark cycle and fed ad libitum with Purina normal rat chow, diet 5001. Groups of 4 Sprague-Dawley rats each were injected subcutaneously with various doses of ISIS 703802 as described in the Table below for 6 weeks (on days 1, 3, 5, 8, 14, 21, 28, 35, and 42). One group of 4 rats was injected subcutaneously with PBS for 6 weeks. Rats were euthanized 48 hours after the last dose, and organs and plasma were harvested for further analysis.

Liver and Kidney Function

To evaluate the effect of ISIS 703802 on hepatic function, plasma levels of transaminases were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.). Plasma levels of ALT (alanine transaminase) and AST (aspartate transaminase) were measured on day 44 and the results are presented in the Table below expressed in IU/L.

To evaluate the effect of ISIS 703802 on renal function, plasma levels of albumin, blood urea nitrogen (BUN), creatitine and bilirubin were measured using the same clinical chemistry analyzer and the results are presented in the Table below expressed in g/dL or mg/dL.

To further evaluate the effect of ISIS 703802 on renal function, urine protein and urine creatinine levels were measured, and the ratio of total urine protein to creatinine was evaluated. The results are presented in the Table below.

ISIS 703802 was shown to be a tolerable compound even at high doses.

TABLE 237 Liver and kidney function markers in Sprague-Dawley rat plasma on day 44 ALT AST Albumin BUN Creatinine Bilurubin (IU/L) (IU/L) (g/dL) (mg/dL) (mg/dL) (mg/dL) PBS 28 72 3.2 15 0.25 0.07 ISIS 703802 86 97 3.4 17 0.26 0.09 50 mg/kg/wk ISIS 703802 62 91 3.3 18 0.29 0.09 20 mg/kg/wk ISIS 703802 64 99 3.2 15 0.27 0.08 10 mg/kg/wk ISIS 703802 48 88 3.3 15 0.26 0.07  5 mg/kg/wk

TABLE 238 Kidney function urine markers (mg/dL) in Sprague-Dawley rat on day 44 Creatinine MTP Protein:Creatinine (mg/dL) (mg/dL) ratio PBS 91 100 1.13 ISIS 703802 82 172 2.04 50 mg/kg/wk ISIS 703802 89 178 2.05 20 mg/kg/wk ISIS 703802 85 103 1.26 10 mg/kg/wk ISIS 703802 117 134 1.17  5 mg/kg/wk Organ Weights

Body, liver, spleen and kidney weights were measured at the end of the study on day 44 and are presented in the Table below. ISIS 703802 did not significantly change body, kidney and liver weights even when administered at high doses.

TABLE 239 Body and organ weights (g) of Sprague Dawley rats Body Kidney Liver Spleen PBS 471 3.6 13 0.67 ISIS 703802 445 3.6 14 1.37 50 mg/kg/wk ISIS 703802 435 3.3 14 0.97 20 mg/kg/wk ISIS 703802 464 3.5 14 0.91 10 mg/kg/wk ISIS 703802 468 3.0 15 0.75  5 mg/kg/wk 

The invention claimed is:
 1. A compound, comprising a single stranded modified oligonucleotide and a conjugate group, wherein the modified oligonucleotide consists of 12 to 30 linked nucleosides and wherein the nucleobase sequence of the modified oligonucleotide comprises at least 8 contiguous nucleobases complementary to an equal length portion of nucleobases 1140 to 1159 of SEQ ID NO: 1, wherein the nucleobase sequence of the modified oligonucleotide is at least 80% complementary to SEQ ID NO: 1; wherein the conjugate group is linked to the modified oligonucleotide at the 5′ end of the modified oligonucleotide by a conjugate linker; and wherein the conjugate group comprises:

or a salt thereof.
 2. The compound or salt of claim 1, wherein the nucleobase sequence of the modified oligonucleotide is at least 85% complementary to SEQ ID NO:
 1. 3. The compound or salt of claim 1, wherein the modified oligonucleotide comprises at least one modified internucleoside linkage.
 4. The compound or salt of claim 3, wherein the at least one modified internucleoside linkage is a phosphorothioate internucleoside linkage.
 5. The compound or salt of claim 1, wherein the modified oligonucleotide comprises at least one modified sugar.
 6. The compound or salt of claim 5, wherein the at least one modified sugar is selected from the group consisting of a bicyclic sugar, a 2′-O-methoxyethyl modified sugar, a constrained ethyl modified sugar, a 3′-fluoro-HNA, and a 4′-(CH₂)_(n)—O-2′ bridge, wherein n is 1 or
 2. 7. The compound or salt of claim 1, wherein at least one nucleoside comprises a modified nucleobase.
 8. The compound or salt of claim 7, wherein the modified nucleobase is a 5-methylcytosine.
 9. The compound or salt of claim 1, wherein the modified oligonucleotide comprises: a gap segment consisting of linked deoxynucleosides; a 5′ wing segment consisting of linked nucleosides; a 3′ wing segment consisting of linked nucleosides; wherein the gap segment is positioned between the 5′ wing segment and the 3′ wing segment and wherein each nucleoside of each wing segment comprises a modified sugar.
 10. The compound or salt claim 1, wherein the modified oligonucleotide comprises: a gap segment consisting of ten linked deoxynucleosides; a 5′ wing segment consisting of five linked nucleosides; a 3′ wing segment consisting of five linked nucleosides; wherein the gap segment is positioned between the 5′ wing segment and the 3′ wing segment, wherein each nucleoside of each wing segment comprises a 2′-O-methoxyethyl sugar, and wherein each cytosine residue is a 5-methylcytosine.
 11. The compound or salt of claim 1, wherein each internucleoside linkage of the modified oligonucleotide is a phosphorothioate linkage.
 12. The compound or salt of claim 10, wherein the modified oligonucleotide consists of 20 linked nucleosides.
 13. The compound or salt of claim 1, wherein the conjugate group comprises a cleavable moiety selected from the group consisting of a phosphodiester, an amide, and an ester.
 14. A pharmaceutical composition comprising the compound or salt of claim 1 and a pharmaceutically acceptable carrier or diluent.
 15. A method for treating a disease, disorder, or condition related to elevated ANGPTL3 in a human in need thereof, comprising administering to the human a therapeutically effective amount of the compound or salt of claim 1, wherein the disease, disorder, or condition is a cardiovascular and/or metabolic disease, disorder, or condition.
 16. The method of claim 15, wherein the disease, disorder or condition is selected from the group consisting of atherosclerosis, hepatic steatosis, obesity, diabetes, dyslipidemia, coronary heart disease, non-alcoholic fatty liver disease (NAFLD), hyperfattyacidemia, and metabolic syndrome.
 17. The method of claim 16, wherein the disease, disorder, or condition is hepatic steatosis and wherein the hepatic steatosis is nonalcoholic steatohepatitis (NASH).
 18. The method of claim 16, wherein the disease, disorder, or condition is dyslipidemia, wherein the dyslipidemia is selected from the group consisting of hyperlipidemia, hypercholesterolemia, and hypertriglyceridemia.
 19. The compound or salt of claim 1, wherein the nucleobase sequence of the modified oligonucleotide is at least 90% complementary to SEQ ID NO:
 1. 20. The compound or salt of claim 1, wherein the nucleobase sequence of the modified oligonucleotide is at least 95% complementary to SEQ ID NO:
 1. 21. The compound or salt of claim 1, wherein the nucleobase sequence of the modified oligonucleotide is 100% complementary to SEQ ID NO:
 1. 22. The method of claim 15, wherein the compound or salt is present in a pharmaceutically acceptable composition. 